RNA-guided nucleases and active fragments and variants thereof and methods of use

Abstract

Compositions and methods for binding to a target sequence of interest are provided. The compositions find use in cleaving or modifying a target sequence of interest, visualization of a target sequence of interest, and modifying the expression of a sequence of interest. Compositions comprise RNA-guided nuclease (RGN) polypeptides, CRISPR RNAs, guide RNAs, and nucleic acid molecules encoding the same. Vectors and host cells comprising the nucleic acid molecules are also provided. Further provided are RGN systems for binding a target sequence of interest, wherein the RGN system comprises an RNA-guided nuclease polypeptide and one or more guide RNAs.

Description

[0001] RNA-GUIDED NUCLEASES AND ACTIVE FRAGMENTS AND VARIANTS THEREOF AND METHODS OF USE

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 63 / 591,255, filed October 18, 2023, and to U.S. Provisional Application No. 63 / 703,673, filed October 4, 2024, each of which is incorporated by referenced herein in its entirety.

[0004] REFERENCE TO A SEQUENCE LISTING SUBMITTED ELECTRONICALLY AS AN XML FILE The instant application contains a Sequence Listing which has been submitted in xml format and is hereby incorporated by reference in its entirety. Said xml copy, created on October 10, 2024, is named L103438_1370WO_0313_1_SL, and is 3,630,310 bytes in size.

[0005] FIELD OF THE INVENTION

[0006] The present invention relates to the field of molecular biology and gene editing.

[0007] BACKGROUND OF THE INVENTION

[0008] Targeted genome editing or modification is rapidly becoming an important tool for basic and applied research. Initial methods involved engineering nucleases such as meganucleases, zinc finger fusion proteins or TALENs, requiring the generation of chimeric nucleases with engineered, programmable, sequencespecific DNA-binding domains specific for each particular target sequence. RNA-guided nucleases, such as the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated (Cas) proteins of the CRISPR-Cas bacterial system, allow for the targeting of specific sequences by complexing the nucleases with guide RNA that specifically hybridizes with a particular target sequence. Producing target-specific guide RNAs is less costly and more efficient than generating chimeric nucleases for each target sequence. Such RNA-guided nucleases can be used to edit genomes optionally through the introduction of a sequencespecific, double-stranded break that is repaired via error-prone non-homologous end-joining (NHEJ) to introduce a mutation at a specific genomic location. Alternatively, heterologous DNA may be introduced into the genomic site via homology-directed repair. RNA-guided nucleases (RGNs) can also be used for base editing when fused with a deaminase.

[0009] BRIEF SUMMARY OF THE INVENTION

[0010] Compositions and methods for binding a target sequence of interest in a target nucleic acid molecule are provided. The compositions find use in cleaving or modifying a target nucleic acid molecule of interest, detection of a target sequence of interest, and modifying the expression of a gene of interest comprising a target sequence. Compositions comprise RNA-guided nuclease (RGN) polypeptides, CRISPR RNAs (crRNAs), guide RNAs (gRNAs), nucleic acid molecules encoding the same, compositions comprising the same, and vectors and host cells comprising the nucleic acid molecules. Also provided are RGN systems and ribonucleoprotein complexes for binding a target sequence of interest, wherein the RGN system and ribonucleoprotein complex comprises an RNA-guided nuclease polypeptide and one or more guide RNAs, where the RGN system and ribonucleoprotein complex does not require a tracrRNA for activity. Thus, methods disclosed herein are drawn to binding a target sequence of interest in a target nucleic acid molecule, and in some embodiments, cleaving or modifying the target nucleic acid molecule of interest without requiring a tracrRNA for activity. The target nucleic acid molecule of interest can be modified, for example, as a result of non-homologous end joining, homology-directed repair with an introduced donor sequence, or base editing. The RGN polypeptides and RGN systems described herein can bind to a protospacer adjacent motif (PAM) site that is located adjacent to and 5' of a target nucleic acid molecule of interest.

[0011] BRIEF DESCRIPTION OF THE FIGURES

[0012] FIGs. 1A-1D show the structures of the LPG10238 (FIG. 1A), LPG10239 (FIG. IB), LPG10240 (FIG. 1C), and LPG10241 (FIG. ID) guide RNAs (i.e. crRNAs) of the present disclosure.

[0013] FIG. 2 shows the % insertion / deletion (Indel) efficiency for LPG10238, LPG10241, LPG13113, and LPG13115 systems. Gene editing efficiency was determined as described in Example 4.

[0014] FIGs. 3A-3EE show sequence logos for the protospacer adjacent motif (PAM) sites recognized by LPG10238 (having the amino acid sequence set forth as SEQ ID NO: 1) (FIG. 3A), LPG10239 (having the amino acid sequence set forth as SEQ ID NO: 2) (FIG. 3B), LPG10240 (having the amino acid sequence set forth as SEQ ID NO: 3) (FIG. 3C), LPG10241 (having the amino acid sequence set forth as SEQ ID NO: 4) (FIG. 3D), LPG13090 (having the amino acid sequence set forth as SEQ ID NO: 1930) (FIG. 3E), LPG13091 (having the amino acid sequence set forth as SEQ ID NO: 1931) (FIG. 3F), LPG13092 (having the amino acid sequence set forth as SEQ ID NO: 1932) (FIG. 3G), LPG13093 (having the amino acid sequence set forth as SEQ ID NO: 1933) (FIG. 3H), LPG13094 (having the amino acid sequence set forth as SEQ ID NO: 1934) (FIG. 31), LPG13095 (having the amino acid sequence set forth as SEQ ID NO: 1935) (FIG. 31), LPG13096 (having the amino acid sequence set forth as SEQ ID NO: 1936) (FIG. 3K), LPG13097 (having the amino acid sequence set forth as SEQ ID NO: 1937) (FIG. 3L), LPG13098 (having the amino acid sequence set forth as SEQ ID NO: 1938) (FIG. 3M), LPG13099 (having the amino acid sequence set forth as SEQ ID NO: 1939) (FIG. 3N), LPG13100 (having the amino acid sequence set forth as SEQ ID NO: 1940) (FIG. 30), LPG13101 (having the amino acid sequence set forth as SEQ ID NO: 1941) (FIG. 3P), LPG13102 (having the amino acid sequence set forth as SEQ ID NO: 1942) (FIG. 3Q), LPG13103 (having the amino acid sequence set forth as SEQ ID NO: 1943) (FIG. 3R), LPG13104 (having the amino acid sequence set forth as SEQ ID NO: 1944) (FIG. 3S), LPG13105 (having the amino acid sequence set forth as SEQ ID NO: 1945) (FIG. 3T), LPG13106 (having the amino acid sequence set forth as SEQ ID NO: 1946) (FIG. 3U), LPG13107 (having the amino acid sequence set forth as SEQ ID NO: 1947) (FIG. 3V), LPG13108 (having the amino acid sequence set forth as SEQ ID NO: 1948) (FIG. 3W),

[0015] LPG13109 (having the amino acid sequence set forth as SEQ ID NO: 1949) (FIG. 3X), LPG13110 (having the amino acid sequence set forth as SEQ ID NO: 1950) (FIG. 3Y), LPG13111 (having the amino acid sequence set forth as SEQ ID NO: 1951) (FIG. 3Z), LPG13112 (having the amino acid sequence set forth as SEQ ID NO: 1952) (FIG. 3AA), LPG13113 (having the amino acid sequence set forth as SEQ ID NO: 1953) (FIG. 3BB), LPG13114 (having the amino acid sequence set forth as SEQ ID NO: 1954) (FIG. 3CC), LPG13115 (having the amino acid sequence set forth as SEQ ID NO: 1955) (FIG. 3DD), and LPG13116 (having the amino acid sequence set forth as SEQ ID NO: 1956) (FIG. 3EE). Each position in a PAM sequence has a stack of nucleotides, where the overall height of each stack indicates the sequence conservation at that position (measured in bits), and the height of each nucleotide within a stack reflects the relative frequency of that nucleotide at that position.

[0016] FIGs. 4A and 4B show mapped domain organization of LPG10241 (FIG. 4A) and LPG10238 (FIG. 4B) as described in Example 5. N=amino terminus; C=carboxy terminus; PI=PAM interacting domain; Reel and RecII domains of the recognition lobe; OBD=oligo binding domain; RuvC-I, RuvC-II, and RuvC-III, tripartite RuvC domain of the nuclease lobe; ZF=zinc finger domain; AS=active site. The three acidic active site residues in the RuvC-I, RuvC-II, and RuvC-III domains are indicated for each RGN polypeptide.

[0017] FIGs. 5A-5D show gene editing activity of LPG10238 mutants (FIGs. 5A and 5B) and LPG10241 mutants (FIGs. 5C and 5D) normalized to parental LPG10238 or LPG10241 gene editing activity, respectively. An initial screen (FIG. 5A, LPG10238; and FIG. 5C, LPG10241) assessed editing for two guides and two genomic loci. From the initial screen, the top 40-90 mutants were selected for further testing in validation rounds with further 2 guides for LPG10238 mutants and 1 guide for LPG10241 mutants (FIG. 5B, LPG10238; and FIG. 5D, LPG10241).

[0018] FIGs. 6A and 6B show that LPG10238 (FIG. 6A) and LPG10241 (FIG. 6B) mutated at active site residues in the RuvC domains failed to show editing activity (active site residues are indicated in FIGs. 4A and 4B and described in Example 5).

[0019] FIGs. 7A and 7B show that the cysteine residues coordinating zinc in the ZF domain of LPG10241 conform to the canonical CxxC motif (FIG. 7A) and are important for editing activity. Mutation of a cysteine at position 687 that is not part of the CxxC motif still allows editing activity.

[0020] FIGs. 8A and 8B show average improvement of editing activity for the best performing 16 LPG10238 mutants (FIG. 8A) and for the best performing 15 LPG10241 mutants (FIG. 8B) over the parental LPG10238 and LPG10241 RGN polypeptides, respectively. The 6 mutants indicated by check marks for each of LPG10238 and LPG 10241 were selected for combinatorial mutant assessment.

[0021] FIGs. 9A and 9B show editing activity for LPG10238 (FIG. 9A) and LPG10241 (FIG. 9B) combinatorial mutants normalized to the parental LPG10238 and LPG10241 activity, respectively. The combinatorial mutants included all possible 63 combinations (6 single mutants, 15 double mutants, 20 triple mutants, 15 quadruple mutants, 6 quintuple mutants, and 1 sextuple mutant) of each selected 6 mutants from FIGs. 8 A and 8B, along with other combinatorial mutants as described in Example 7. The combinatorial mutants were screened with an additional panel of five to six guides. FIGs. 10A and 1OB show percent editing efficiency for the 18 best performing LPG10238 (FIG. 10A) and 17 best performing LPG10241 (FIG. 10B) combinatorial mutants. On average, the top 18 LPG10238 mutants exhibit greater than or equal to 2-fold enhanced editing as compared to parental LPG10238 (FIG. 10A). Similarly, the top 17 LPG 10241 combinatorial mutants demonstrated greater than 1.3-fold editing relative to the parental LPG10241 editing (FIG. 10B). The average percent editing efficiencies across the 5 guide RNAs and for each guide RNA are shown in FIG. 10A. The average percent editing efficiencies for each of the 6 guide RNAs are shown in FIG. 10B. FIG. 10B parental LPG10241 controls include parental LPG 10241 with a parental guide (indicated by circle on x-axis) and parental LPG 10241 with an engineered guide (indicated by ‘WT’ on the x-axis). The percent editing efficiencies indicated above the ‘WT’ bars are, from left to right, 3.8%, 1.3%, 2.9%, 3.6%, 4.9%, and 17.7%. The LPG 10241 combinatorial mutants were tested with engineered guide RNAs.

[0022] FIGs. 11A and 11B show improved editing of LPG10241 mutants as compared to parental LPG 10241. (FIG. 11A) Complete deletion of the C-terminal tail improved activity relative to the parental LPG10241 (FIG. 11A, ‘C-terDell’). The deletion was of amino acids 751-769 (GQTPDSDEESEVALHTATA (SEQ ID NO: 1927)). Mutation of the C-terminal tail to a Type V-j consensus (‘Con.Cterl’, 8 arginine or lysine mutations) also improved editing. Finally, a combinatorial mutant combining 8 single arginine mutations from the C-terminal region improved editing greater than 2x relative to the parental (‘R.ScanCter’). (FIG. 11B) Mutation of the zinc finger domain (i.e. zinc binding domain) to the consensus improved editing greater than 2x (‘Cons.Znl’), while combining four of the single-arginine mutations improved editing to near 4x (‘R.ScanZnl’).

[0023] FIGs. 12A-12D show percent editing efficiencies for the top 3 combinatorial mutants of LPG10238 against a panel of 43 guides (FIGs . 12A and 12B) and for the top 3 combinatorial mutants of LPG 10241 against a panel of 37 guides (FIGs. 12C and 12D).

[0024] FIGs. 13A and 13B show strategies for generating shortened backbone variants of LPG10241 guides (FIG. 13A) and percent editing (NHEJ=non-homologous end joining) for shortened LPG10241 guides as compared to that of parental LPG10241 guide using 3 different spacers, as described in Example 8 and Table 15. The * indicates a shortened crRNA bb. The arrows in FIG. 13A depict the direction in which the shortening of the crRNA bb proceeds, i.e., nucleotides are deleted from the 5’ end of the crRNA bb. The ‘WT’ control in FIG. 13B has an LPG10241 guide of crRNA bb-spacer-cRNA bb (where the crRNA bb is not shortened).

[0025] FIG. 14 show percent editing for shortened LPG10238 guides as compared to that of parental LPG10238 guide using 2 different spacers. Shortening designs for LPG10238 guides are described in Example 8 and Table 16. The crRNA backbone for LPG 10238 could not be shortened to maintain or enhance the editing efficiency of the parental 36 nt backbone. A schematic of the parental LPG 10238 backbone is to the left of the graphs. * crRNA bb = shortened crRNA backbone; S = spacer. FIG. 15 shows average percent editing for LPG10238 guide variants with mutations in the backbone as compared to that of parental LPG10238 guide using 2 different spacers. Backbone variant 2 showed promising improvements in editing over the parental.

[0026] FIGs. 16A-16D show editing activity of LPG10241 (FIGs. 16A and 16B) and LPG10238 (FIGs. 16C and 16D) glycine mutants normalized to parental LPG 10241 and LPG10238 editing activity, respectively. An initial screen (FIGs. 16A and 16C) assessed editing for two guides. Validation screens tested a further two guides (FIGs. 16B and 16D).

[0027] FIGs. 17A and 17B show sequence alignments of the highest frequency excision amplicons for LDHA target (FIG. 17A) and B2M target (FIG. 17B) to the respective parental sequences. Guide target sites are shown as arrows. Excision is with LPG10238 systems described in Example 11.

[0028] FIGs. 18A and 18B show genomic excision activity of a mutant LPG10241, LPG13264. (FIG. 18A) Agarose gel of repair product amplicons. A and B are from single guide edits where no excision is anticipated, while samples C and D are from excision edits. (-) lanes have no guides. (FIG. 18B) Frequency distribution of the deletion size of excision products. Excision is with LPG 10241 systems described in Example 11.

[0029] FIGs. 19A and 19B. (FIG. 19A) In vitro cleavage progress curves of LPG10238 using a synthetic tandem guide array targeting 2 double-stranded DNA (dsDNA) substrates (L3, L2). (FIG. 19B) In vitro cleavage progress curves of LPG 10238 using a mixture of 2 synthetic single guides each targeting a dsDNA substrate (L3, L2), as described in Example 12. NTS=non-target strand; TS=target strand.

[0030] FIGs. 20A and 20B. (FIG. 20A) In vitro cleavage progress curves of LPG10241 using a synthetic tandem guide array targeting 2 dsDNA substrates (L3, L2). (FIG. 20B) In vitro cleavage progress curves of LPG10241 using a mixture of 2 synthetic single guides each targeting a dsDNA substrate (L3, L2), as described in Example 12. NTS=non-target strand; TS=target strand.

[0031] FIGs. 21A and 21B. (FIG. 21A) Guide processing curves for active LPG10238 on two synthetic guide arrays (circle, square), and catalytically inactive LPG10238 on the same arrays (triangle, inverted triangle). (FIG. 2 IB) Guide processing curves for active LPG 10241 on two synthetic guide arrays (circle, square), and catalytically inactive LPG10241 on the same arrays (triangle, inverted triangle).

[0032] FIGs. 22A and 22B show all-in-one AAV plasmid delivery to HEK293T cells. Editing at B2M (FIG. 22A) and at LDHA (FIG. 22B) are shown for LPG 10238 systems.

[0033] FIGs. 23A and 23B show cut-site characterization summary for LPG10241 and LPG10238, as described in Example 14. (FIG. 23A) cleavage site charaterization for target 1 (LI), and (FIG. 23B) cleavage site charaterization for target 2 (L2).

[0034] DETAILED DESCRIPTION

[0035] Many modifications and other embodiments of the inventions set forth herein will come to mind to one skilled in the art to which these inventions pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the inventions are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended embodiments. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0036] I. Overview

[0037] RNA-guided nucleases (RGNs) allow for the targeted manipulation of specific site(s) within a genome and are useful in the context of gene targeting for therapeutic and research applications. In a variety of organisms, including mammals, RGNs have been used for genome engineering by stimulating non- homologous end joining and homologous recombination, for example.

[0038] In particular, in various embodiments, the RGNs and systems (e.g., an RGN and a corresponding gRNA) of the disclosure include Type V-H Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) enzymes and Type V-J Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) enzymes. The Type V-H’s and Type V-J’s described herein are particularly interesting at least due to their smaller size (-900 amino acids or less) and atypical protospacer adjacent motif (PAM) sequences compared to most characterized Type II CRISPR enzymes. The Type V-H systems described herein utilize AT-rich PAMs 5’ to target sequences as compared to Type II systems, which utilize PAMs 3’ of the target sequences. Similarly, the Type V-J systems described herein utilize T-rich PAMs 5’ to target sequences. The smaller size of Type V nucleases allows delivery of these enzymes by adeno-associated viral (AAV) vectors used in preclinical and clinical settings. Additionally, because the Type V systems described herein only utilize a CRISPR RNA (crRNA) without need of a transactivating CRISPR RNA (tracrRNA), their associated guide RNAs are compact (about 40 nucleotides or less) relative to Type II guide RNAs. The smaller guide RNAs of the Type V editing systems described herein allow easier manufacturing of the guide RNA with higher purity. In various embodiments, the Type V enzymes described herein generate overhang (i.e. staggered) cuts in a nucleic acid molecule, which is well suited for insertions of donor polynucleotides. Importantly, because the Type V guide RNAs described herein are self-processing and relatively short in length, one can take advantage of these features to create constructs with a tandem arrangement of multiple Type V guide RNAs for editing of multiple targets. The Type V systems described herein also function in mammalian cells, which has not yet been demonstrated in the literature for the Type V-H’s. Besides their biochemical characteristics, the Type V-H subtype is of particular interest due to its relatively rarity across sequenced genomes. With its CRISPR loci being more challenging to annotate using traditional CRISPR annotation pipelines, the subtype is poorly described in the literature, with a single publicly known example (Y an et al. (2019) Science 363(6422): 88-91), which utilizes RTR PAMs (and in particular TG-rich PAMs) for dsDNA binding and for which editing activity has not been reported as far as the inventors are aware. Thus, the highly active Type V-H enzymes and systems described herein represent a significant contribution to the field. The compositions and methods described herein are useful for creating breaks in polynucleotides, modifying polynucleotides, detecting a particular site within a polynucleotide, or modifying the expression of a particular gene. In particular embodimenst, the compositions and methods described herein are useful for creating double-stranded breaks in polynucleotides, modifying polynucleotides, detecting a particular site within a polynucleotide, or modifying the expression of a particular gene.

[0039] The RGNs disclosed herein can alter gene expression by modifying a target nucleic acid molecule comprising a target sequence. In specific embodiments, the RGNs are directed to a target sequence (e.g., target DNA sequence) by a guide RNA (gRNA) as part of a CRISPR RGN system. The RGNs are considered “RNA-guided” because guide RNAs form a complex with the RGNs to direct the RGN to bind to a target sequence and in some embodiments, introduce a double-stranded break at the target sequence (e.g., target DNA sequence). After the target sequence has been cleaved, the break can be repaired such that the sequence of the target nucleic acid molecule is modified during the repair process. Thus, provided herein are methods for using the RGNs to modify a target nucleic acid molecule in a host cell. For example, RGNs can be used to modify a target sequence at a genomic locus of eukaryotic cells or prokaryotic cells.

[0040] II. RNA-guided nucleases

[0041] Provided herein are RNA-guided nucleases. The term RNA-guided nuclease (RGN) refers to a polypeptide that binds to a particular target sequence (e.g., target DNA sequence) in a sequence-specific manner and is directed to the target sequence by a guide RNA molecule that is complexed with the polypeptide and hybridizes with the target sequence. Although an RGN can be capable of cleaving the target sequence upon binding, the term RGN also encompasses nuclease-dead RGNs that are capable of binding to, but not cleaving, a target sequence. Cleavage of a target sequence by an RGN can result in a single- or double-stranded break. RGNs only capable of cleaving a single strand of a double-stranded target nucleic acid molecule are referred to herein as nickases.

[0042] The RNA-guided nucleases disclosed herein include the LPG10238, LPG10239, LPG10240, LPG10241, LPG13090, LPG13091, LPG13092, LPG13093, LPG13094, LPG13095, LPG13096, LPG13097, LPG13098, LPG13099, LPG13100, LPG13101, LPG13102, LPG13103, LPG13104, LPG13105, LPG13106, LPG13107, LPG13108, LPG13109, LPG13110, LPG13111, LPG13112, LPG13113, LPG13114, LPG13115, and LPG13116 RGNs, the amino acid sequences of which are set forth, respectively, as SEQ ID NOs: 1-4 and 1930-1956, and active fragments or variants thereof that retain the ability to bind to a target sequence in an RNA-guided sequence-specific manner. In some of these embodiments, the active fragment or variant of the LPG10238, LPG10239, LPG10240, LPG10241, LPG13090, LPG13091, LPG13092, LPG13093, LPG13094, LPG13095, LPG13096, LPG13097, LPG13098, LPG13099, LPG13100, LPG13101, LPG13102, LPG13103, LPG13104, LPG13105, LPG13106, LPG13107, LPG13108, LPG13109, LPG13110, LPG13111, LPG13112, LPG13113, LPG13114, LPG13115, or LPG13116 RGN is capable of cleaving a double-stranded target sequence. In some embodiments, an active variant of the LPG10238, LPG10239, LPG10240, LPG10241, LPG13090, LPG13091, LPG13092, LPG13093, LPG13094, LPG13095, LPG13096, LPG13097, LPG13098, LPG13099, LPG13100, LPG13101, LPG13102, LPG13103, LPG13104, LPG13105, LPG13106, LPG13107, LPG13108, LPG13109, LPG13110, LPG13111, LPG13112, LPG13113, LPG13114, LPG13115, or LPG13116 RGN comprises an amino acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence set forth as any one of SEQ ID NOs: 1-4 and 1930-1956.

[0043] In some embodiments, an active variant of the LPG10238 RGN comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 and retains RNA-guided sequence-specific binding activity. In some embodiments, an active variant of the LPG10238 RGN comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 and retains RNA-guided sequence -specific binding activity. In some embodiments, an active variant of the LPG10238 RGN comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 and retains RNA-guided sequence -specific binding activity. In some embodiments, an active variant of the LPG10238 RGN comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 and retains RNA-guided sequence-specific binding activity.

[0044] In some embodiments, an active variant of the LPG10239 RGN comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2 and retains RNA-guided sequence-specific binding activity. In some embodiments, an active variant of the LPG10239 RGN comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2 and retains RNA-guided sequence -specific binding activity. In some embodiments, an active variant of the LPG10239 RGN comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2 and retains RNA-guided sequence -specific binding activity. In some embodiments, an active variant of the LPG10239 RGN comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2 and retains RNA-guided sequence-specific binding activity.

[0045] In some embodiments, an active variant of the LPG 10240 RGN comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3 and retains RNA-guided sequence-specific binding activity. In some embodiments, an active variant of the LPG10240 RGN comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3 and retains RNA-guided sequence -specific binding activity. In some embodiments, an active variant of the LPG 10240 RGN comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3 and retains RNA-guided sequence -specific binding activity. In some embodiments, an active variant of the LPG10240 RGN comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3 and retains RNA-guided sequence-specific binding activity.

[0046] In some embodiments, an active variant of the LPG 10241 RGN comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4 and retains RNA-guided sequence-specific binding activity. In some embodiments, an active variant of the LPG10241 RGN comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4 and retains RNA-guided sequence -specific binding activity. In some embodiments, an active variant of the LPG 10241 RGN comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4 and retains RNA-guided sequence -specific binding activity. In some embodiments, an active variant of the LPG10241 RGN comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4 and retains RNA-guided sequence-specific binding activity.

[0047] In some embodiments, an active variant of the LPG 13090 RGN comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1930 and retains RNA-guided sequence-specific binding activity. In some embodiments, an active variant of the LPG13090 RGN comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1930 and retains RNA-guided sequence-specific binding activity. In some embodiments, an active variant of the LPG 13090 RGN comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1930 and retains RNA-guided sequence -specific binding activity. In some embodiments, an active variant of the LPG13090 RGN comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1930 and retains RNA-guided sequence -specific binding activity.

[0048] In some embodiments, an active variant of the LPG 13091 RGN comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1931 and retains RNA-guided sequence-specific binding activity. In some embodiments, an active variant of the LPG13091 RGN comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1931 and retains RNA-guided sequence-specific binding activity. In some embodiments, an active variant of the LPG 13091 RGN comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1931 and retains RNA-guided sequence -specific binding activity. In some embodiments, an active variant of the LPG13091 RGN comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1931 and retains RNA-guided sequence -specific binding activity.

[0049] In some embodiments, an active variant of the LPG 13092 RGN comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1932 and retains RNA-guided sequence-specific binding activity. In some embodiments, an active variant of the LPG13092 RGN comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1932 and retains RNA-guided sequence-specific binding activity. In some embodiments, an active variant of the LPG13092 RGN comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1932 and retains RNA-guided sequence -specific binding activity. In some embodiments, an active variant of the LPG13092 RGN comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1932 and retains RNA-guided sequence -specific binding activity.

[0050] In some embodiments, an active variant of the LPG13093 RGN comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1933 and retains RNA-guided sequence-specific binding activity. In some embodiments, an active variant of the LPG13093 RGN comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1933 and retains RNA-guided sequence-specific binding activity. In some embodiments, an active variant of the LPG13093 RGN comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1933 and retains RNA-guided sequence -specific binding activity. In some embodiments, an active variant of the LPG13093 RGN comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1933 and retains RNA-guided sequence -specific binding activity.

[0051] In some embodiments, an active variant of the LPG13094 RGN comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1934 and retains RNA-guided sequence-specific binding activity. In some embodiments, an active variant of the LPG13094 RGN comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1934 and retains RNA-guided sequence-specific binding activity. In some embodiments, an active variant of the LPG13094 RGN comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1934 and retains RNA-guided sequence -specific binding activity. In some embodiments, an active variant of the LPG13094 RGN comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1934 and retains RNA-guided sequence -specific binding activity.

[0052] In some embodiments, an active variant of the LPG13095 RGN comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1935 and retains RNA-guided sequence-specific binding activity. In some embodiments, an active variant of the LPG13095 RGN comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1935 and retains RNA-guided sequence-specific binding activity. In some embodiments, an active variant of the LPG13095 RGN comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1935 and retains RNA-guided sequence -specific binding activity. In some embodiments, an active variant of the LPG13095 RGN comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1935 and retains RNA-guided sequence -specific binding activity.

[0053] In some embodiments, an active variant of the LPG13096 RGN comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1936 and retains RNA-guided sequence-specific binding activity. In some embodiments, an active variant of the LPG13096 RGN comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1936 and retains RNA-guided sequence-specific binding activity. In some embodiments, an active variant of the LPG13096 RGN comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1936 and retains RNA-guided sequence -specific binding activity. In some embodiments, an active variant of the LPG13096 RGN comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1936 and retains RNA-guided sequence -specific binding activity.

[0054] In some embodiments, an active variant of the LPG13097 RGN comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1937 and retains RNA-guided sequence-specific binding activity. In some embodiments, an active variant of the LPG13097 RGN comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1937 and retains RNA-guided sequence-specific binding activity. In some embodiments, an active variant of the LPG13097 RGN comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1937 and retains RNA-guided sequence -specific binding activity. In some embodiments, an active variant of the LPG13097 RGN comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1937 and retains RNA-guided sequence -specific binding activity.

[0055] In some embodiments, an active variant of the LPG13098 RGN comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1938 and retains RNA-guided sequence-specific binding activity. In some embodiments, an active variant of the LPG13098 RGN comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1938 and retains RNA-guided sequence-specific binding activity. In some embodiments, an active variant of the LPG13098 RGN comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1938 and retains RNA-guided sequence -specific binding activity. In some embodiments, an active variant of the LPG13098 RGN comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1938 and retains RNA-guided sequence -specific binding activity.

[0056] In some embodiments, an active variant of the LPG13099 RGN comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1939 and retains RNA-guided sequence-specific binding activity. In some embodiments, an active variant of the LPG13099 RGN comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1939 and retains RNA-guided sequence-specific binding activity. In some embodiments, an active variant of the LPG13099 RGN comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1939 and retains RNA-guided sequence -specific binding activity. In some embodiments, an active variant of the LPG13099 RGN comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1939 and retains RNA-guided sequence -specific binding activity.

[0057] In some embodiments, an active variant of the LPG13100 RGN comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1940 and retains RNA-guided sequence-specific binding activity. In some embodiments, an active variant of the LPG13100 RGN comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1940 and retains RNA-guided sequence-specific binding activity. In some embodiments, an active variant of the LPG13100 RGN comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1940 and retains RNA-guided sequence -specific binding activity. In some embodiments, an active variant of the LPG13100 RGN comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1940 and retains RNA-guided sequence -specific binding activity.

[0058] In some embodiments, an active variant of the LPG13101 RGN comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1941 and retains RNA-guided sequence-specific binding activity. In some embodiments, an active variant of the LPG13101 RGN comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1941 and retains RNA-guided sequence-specific binding activity. In some embodiments, an active variant of the LPG13101 RGN comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1941 and retains RNA-guided sequence -specific binding activity. In some embodiments, an active variant of the LPG13101 RGN comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1941 and retains RNA-guided sequence -specific binding activity.

[0059] In some embodiments, an active variant of the LPG13102 RGN comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1942 and retains RNA-guided sequence-specific binding activity. In some embodiments, an active variant of the LPG13102 RGN comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1942 and retains RNA-guided sequence-specific binding activity. In some embodiments, an active variant of the LPG13102 RGN comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1942 and retains RNA-guided sequence -specific binding activity. In some embodiments, an active variant of the LPG13102 RGN comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1942 and retains RNA-guided sequence -specific binding activity.

[0060] In some embodiments, an active variant of the LPG13103 RGN comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1943 and retains RNA-guided sequence-specific binding activity. In some embodiments, an active variant of the LPG13103 RGN comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1943 and retains RNA-guided sequence-specific binding activity. In some embodiments, an active variant of the LPG13103 RGN comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1943 and retains RNA-guided sequence -specific binding activity. In some embodiments, an active variant of the LPG13103 RGN comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1943 and retains RNA-guided sequence -specific binding activity.

[0061] In some embodiments, an active variant of the LPG13104 RGN comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1944 and retains RNA-guided sequence-specific binding activity. In some embodiments, an active variant of the LPG13104 RGN comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1944 and retains RNA-guided sequence-specific binding activity. In some embodiments, an active variant of the LPG13104 RGN comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1944 and retains RNA-guided sequence -specific binding activity. In some embodiments, an active variant of the LPG13104 RGN comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1944 and retains RNA-guided sequence -specific binding activity.

[0062] In some embodiments, an active variant of the LPG13105 RGN comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1945 and retains RNA-guided sequence-specific binding activity. In some embodiments, an active variant of the LPG13105 RGN comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1945 and retains RNA-guided sequence-specific binding activity. In some embodiments, an active variant of the LPG13105 RGN comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1945 and retains RNA-guided sequence -specific binding activity. In some embodiments, an active variant of the LPG13105 RGN comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1945 and retains RNA-guided sequence -specific binding activity.

[0063] In some embodiments, an active variant of the LPG13106 RGN comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1946 and retains RNA-guided sequence-specific binding activity. In some embodiments, an active variant of the LPG13106 RGN comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1946 and retains RNA-guided sequence-specific binding activity. In some embodiments, an active variant of the LPG13106 RGN comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1946 and retains RNA-guided sequence -specific binding activity. In some embodiments, an active variant of the LPG13106 RGN comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1946 and retains RNA-guided sequence -specific binding activity.

[0064] In some embodiments, an active variant of the LPG13107 RGN comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1947 and retains RNA-guided sequence-specific binding activity. In some embodiments, an active variant of the LPG13107 RGN comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1947 and retains RNA-guided sequence-specific binding activity. In some embodiments, an active variant of the LPG13107 RGN comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1947 and retains RNA-guided sequence -specific binding activity. In some embodiments, an active variant of the LPG13107 RGN comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1947 and retains RNA-guided sequence -specific binding activity.

[0065] In some embodiments, an active variant of the LPG13108 RGN comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1948 and retains RNA-guided sequence-specific binding activity. In some embodiments, an active variant of the LPG13108 RGN comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1948 and retains RNA-guided sequence-specific binding activity. In some embodiments, an active variant of the LPG13108 RGN comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1948 and retains RNA-guided sequence -specific binding activity. In some embodiments, an active variant of the LPG13108 RGN comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1948 and retains RNA-guided sequence -specific binding activity.

[0066] In some embodiments, an active variant of the LPG13109 RGN comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1949 and retains RNA-guided sequence-specific binding activity. In some embodiments, an active variant of the LPG13109 RGN comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1949 and retains RNA-guided sequence-specific binding activity. In some embodiments, an active variant of the LPG13109 RGN comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1949 and retains RNA-guided sequence -specific binding activity. In some embodiments, an active variant of the LPG13109 RGN comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1949 and retains RNA-guided sequence -specific binding activity.

[0067] In some embodiments, an active variant of the LPG13110 RGN comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1950 and retains RNA-guided sequence-specific binding activity. In some embodiments, an active variant of the LPG13110 RGN comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1950 and retains RNA-guided sequence-specific binding activity. In some embodiments, an active variant of the LPG13110 RGN comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1950 and retains RNA-guided sequence -specific binding activity. In some embodiments, an active variant of the LPG13110 RGN comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1950 and retains RNA-guided sequence -specific binding activity.

[0068] In some embodiments, an active variant of the LPG13111 RGN comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1951 and retains RNA-guided sequence-specific binding activity. In some embodiments, an active variant of the LPG13111 RGN comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1951 and retains RNA-guided sequence-specific binding activity. In some embodiments, an active variant of the LPG13111 RGN comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1951 and retains RNA-guided sequence -specific binding activity. In some embodiments, an active variant of the LPG13111 RGN comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1951 and retains RNA-guided sequence -specific binding activity.

[0069] In some embodiments, an active variant of the LPG13112 RGN comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1952 and retains RNA-guided sequence-specific binding activity. In some embodiments, an active variant of the LPG13112 RGN comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1952 and retains RNA-guided sequence-specific binding activity. In some embodiments, an active variant of the LPG13112 RGN comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1952 and retains RNA-guided sequence -specific binding activity. In some embodiments, an active variant of the LPG13112 RGN comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1952 and retains RNA-guided sequence -specific binding activity.

[0070] In some embodiments, an active variant of the LPG13113 RGN comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1953 and retains RNA-guided sequence-specific binding activity. In some embodiments, an active variant of the LPG13113 RGN comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1953 and retains RNA-guided sequence-specific binding activity. In some embodiments, an active variant of the LPG13113 RGN comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1953 and retains RNA-guided sequence -specific binding activity. In some embodiments, an active variant of the LPG13113 RGN comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1953 and retains RNA-guided sequence -specific binding activity.

[0071] In some embodiments, an active variant of the LPG13114 RGN comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1954 and retains RNA-guided sequence-specific binding activity. In some embodiments, an active variant of the LPG13114 RGN comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1954 and retains RNA-guided sequence-specific binding activity. In some embodiments, an active variant of the LPG13114 RGN comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1954 and retains RNA-guided sequence -specific binding activity. In some embodiments, an active variant of the LPG13114 RGN comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1954 and retains RNA-guided sequence -specific binding activity.

[0072] In some embodiments, an active variant of the LPG13115 RGN comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1955 and retains RNA-guided sequence-specific binding activity. In some embodiments, an active variant of the LPG13115 RGN comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1955 and retains RNA-guided sequence-specific binding activity. In some embodiments, an active variant of the LPG13115 RGN comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1955 and retains RNA-guided sequence -specific binding activity. In some embodiments, an active variant of the LPG13115 RGN comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1955 and retains RNA-guided sequence -specific binding activity.

[0073] In some embodiments, an active variant of the LPG13116 RGN comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1956 and retains RNA-guided sequence-specific binding activity. In some embodiments, an active variant of the LPG13116 RGN comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1956 and retains RNA-guided sequence-specific binding activity. In some embodiments, an active variant of the LPG13116 RGN comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1956 and retains RNA-guided sequence -specific binding activity. In some embodiments, an active variant of the LPG13116 RGN comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1956 and retains RNA-guided sequence -specific binding activity.

[0074] Retaining or having RNA-guided sequence -specific binding activity refers to an RGN polypeptide having the capability of binding in an RNA-guided sequence-specific manner to a target nucleic acid molecule and can be measured by binding assays known in the art and described herein. In some embodiments, an active variant or fragment of LPG10238, LPG10239, LPG10240, LPG10241, LPG13090, LPG13091, LPG13092, LPG13093, LPG13094, LPG13095, LPG13096, LPG13097, LPG13098, LPG13099, LPG13100, LPG13101, LPG13102, LPG13103, LPG13104, LPG13105, LPG13106, LPG13107, LPG13108, LPG13109, LPG13110, LPG13111, LPG13112, LPG13113, LPG13114, LPG13115, or LPG13116 RGN disclosed herein, or an active variant or fragment thereof, has RNA-guided sequence -specific binding activity that is from about 80% to about 500%, from about 80% to about 200%, or from about 90% to about 150%, or from about 95% to about 120%, or is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 125%, at least 130%, at least 135%, at least 140%, at least 145%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500%, or more, of the RNA-guided sequence -specific binding activity of a reference RGN.

[0075] An RGN polypeptide of the disclosure can retain or have nuclease activity. Nuclease activity can be measured by assays known in the art and described herein. In some embodiments, an active variant or fragment of LPG10238, LPG10239, LPG10240, LPG10241, LPG13090, LPG13091, LPG13092, LPG13093, LPG13094, LPG13095, LPG13096, LPG13097, LPG13098, LPG13099, LPG13100, LPG13101, LPG13102, LPG13103, LPG13104, LPG13105, LPG13106, LPG13107, LPG13108, LPG13109, LPG13110, LPG13111, LPG13112, LPG13113, LPG13114, LPG13115, or LPG13116 RGN disclosed herein, or an active variant or fragment thereof, has nuclease activity that is from about 80% to about 500%, from about 80% to about 200%, or from about 90% to about 150%, or from about 95% to about 120%, or is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 125%, at least 130%, at least 135%, at least 140%, at least 145%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500%, or more, of the nuclease activity of a reference RGN.

[0076] In some embodiments, a reference RGN has the amino acid sequence set forth as any one of SEQ ID NOs: 1-4, and 1930-1956. In some embodiments, a reference RGN is a variant of any one of SEQ ID NOs: 1-4, and 1930-1956 that lacks the corresponding mutations of the active variant or fragment thereof that has RNA-guided sequence-specific binding activity or nuclease activity, e.g., as described above. In some embodiments, a reference RGN is a non-identical variant RGN. In some embodiments, a reference RGN is a non-identical variant RGN sharing the same parental RGN. A ‘parental RGN’ or ‘parental RGN polypeptide’ refers to a parent polypeptide which can function as a template to introduce one or more mutations to generate variant polypeptides of that parent polypeptide. For example, LPG10238 is the parental RGN polypeptide of mutant RGN polypeptides LPG10427 to LPG10821, and LPG12222, as discussed in Example 6. Other parental RGN polypeptides of the disclosure include LPG10239, LPG10240, LPG1024I, LPG13090, LPG1309I, LPG13092, LPG13093, LPG13094, LPG13095, LPG13096, LPG13097, LPG13098, LPG13099, LPG13100, LPG13I0I, LPG13102, LPG13103, LPG13104, LPG13105, LPG13106, LPG13107, LPG13108, LPG13109, LPG13110, LPG13111, LPG13112, LPG13113, LPG13114, LPG13115, and LPG13116.

[0077] An active fragment of the LPG10238 RGN can comprise at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, or more contiguous amino acid residues of the amino acid sequence set forth as SEQ ID NO: 1. An active fragment of the LPG10239 RGN can comprise at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or more contiguous amino acid residues of the amino acid sequence set forth as SEQ ID NO: 2. An active fragment of the LPG10240 RGN can comprise at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or more contiguous amino acid residues of the amino acid sequence set forth as SEQ ID NO: 3. An active fragment of the LPG10241 RGN can comprise at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or more contiguous amino acid residues of the amino acid sequence set forth as SEQ ID NO: 4. An active fragment of the LPG13090 RGN can comprise at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or more contiguous amino acid residues of the amino acid sequence set forth as SEQ ID NO: 1930. An active fragment of the LPG13091 RGN can comprise at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or more contiguous amino acid residues of the amino acid sequence set forth as SEQ ID NO: 1931. An active fragment of the LPG13092 RGN can comprise at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or more contiguous amino acid residues of the amino acid sequence set forth as SEQ ID NO: 1932. An active fragment of the LPG13093 RGN can comprise at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or more contiguous amino acid residues of the amino acid sequence set forth as SEQ ID NO: 1933. An active fragment of the LPG13094 RGN can comprise at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or more contiguous amino acid residues of the amino acid sequence set forth as SEQ ID NO: 1934. An active fragment of the LPG13095 RGN can comprise at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or more contiguous amino acid residues of the amino acid sequence set forth as SEQ ID NO: 1935. An active fragment of the LPG13096 RGN can comprise at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or more contiguous amino acid residues of the amino acid sequence set forth as SEQ ID NO: 1936. An active fragment of the LPG13097 RGN can comprise at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or more contiguous amino acid residues of the amino acid sequence set forth as SEQ ID NO: 1937. An active fragment of the LPG13098 RGN can comprise at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or more contiguous amino acid residues of the amino acid sequence set forth as SEQ ID NO: 1938. An active fragment of the LPG13099 RGN can comprise at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or more contiguous amino acid residues of the amino acid sequence set forth as SEQ ID NO: 1939. An active fragment of the LPG13100 RGN can comprise at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or more contiguous amino acid residues of the amino acid sequence set forth as SEQ ID NO: 1940. An active fragment of the

[0078] LPG13101 RGN can comprise at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or more contiguous amino acid residues of the amino acid sequence set forth as SEQ ID NO: 1941. An active fragment of the LPG13102 RGN can comprise at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or more contiguous amino acid residues of the amino acid sequence set forth as SEQ ID NO: 1942. An active fragment of the LPG13103 RGN can comprise at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or more contiguous amino acid residues of the amino acid sequence set forth as SEQ ID NO: 1943. An active fragment of the LPG13104 RGN can comprise at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or more contiguous amino acid residues of the amino acid sequence set forth as SEQ ID NO: 1944. An active fragment of the LPG13105 RGN can comprise at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or more contiguous amino acid residues of the amino acid sequence set forth as SEQ ID NO: 1945. An active fragment of the LPG13106 RGN can comprise at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or more contiguous amino acid residues of the amino acid sequence set forth as SEQ ID NO: 1946. An active fragment of the LPG13107 RGN can comprise at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or more contiguous amino acid residues of the amino acid sequence set forth as SEQ ID NO: 1947. An active fragment of the LPG13108 RGN can comprise at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or more contiguous amino acid residues of the amino acid sequence set forth as SEQ ID NO: 1948. An active fragment of the LPG13109 RGN can comprise at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or more contiguous amino acid residues of the amino acid sequence set forth as SEQ ID NO: 1949. An active fragment of the LPG13110 RGN can comprise at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or more contiguous amino acid residues of the amino acid sequence set forth as SEQ ID NO: 1950. An active fragment of the LPG13111 RGN can comprise at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or more contiguous amino acid residues of the amino acid sequence set forth as SEQ ID NO: 1951. An active fragment of the LPG13112 RGN can comprise at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or more contiguous amino acid residues of the amino acid sequence set forth as SEQ ID NO: 1952. An active fragment of the LPG13113 RGN can comprise at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or more contiguous amino acid residues of the amino acid sequence set forth as SEQ ID NO: 1953. An active fragment of the LPG13114 RGN can comprise at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or more contiguous amino acid residues of the amino acid sequence set forth as SEQ ID NO: 1954. An active fragment of the LPG13115 RGN can comprise at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or more contiguous amino acid residues of the amino acid sequence set forth as SEQ ID NO: 1955. An active fragment of the LPG13116 RGN can comprise at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or more contiguous amino acid residues of the amino acid sequence set forth as SEQ ID NO: 1956.

[0079] An RGN polypeptide of the disclosure can comprise an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1 (LPG10238), and wherein the amino acid residue(s) at one or more of amino acid positions 517, 530, 538, 548, 551, 552, 553, 568, 570, 574, 577, 611, 677, 789, 790, and 801 of said RGN polypeptide differs from the corresponding amino acid residue of SEQ ID NO: 1. In some embodiments, the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1, and wherein the amino acid residue(s) at one or more of amino acid positions 517, 530, 538, 548, 551, 552, 553, 568, 570, 574, 577, 611, 677, 789, 790, and 801 of said RGN polypeptide is a positively charged amino acid residue. In some embodiments, the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1, and wherein the amino acid residue(s) at one or more of amino acid positions 517, 530, 538, 548, 551, 552, 553, 568, 570, 574, 577, 611, 677, 789, 790, and 801 of said RGN polypeptide is an arginine (R). In some embodiments, the RGN polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1, and wherein the amino acid residue(s) at one or more of amino acid positions 517, 530, 538, 548, 551, 552, 553, 568, 570, 574, 577, 611, 677, 789, 790, and 801 of said RGN polypeptide is an R. An RGN polypeptide of the disclosure can comprise the amino acid sequence set forth as any one of SEQ ID NOs: 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 602, 707, 708, and 717.

[0080] When referring to a variant RGN “wherein the amino acid residue at one or more of amino acid positions. . . of said RGN polypeptide differs from the corresponding amino acid residue of SEQ ID NO: ... ”, it is intended to mean that the variant RGN comprises an amino acid sequence that is identical to the amino acid sequence set forth in the recited SEQ ID NO:, except that amino acid residues at the recited positions differ from the corresponding amino acid residues in the recited SEQ ID NO:. When referring to an amino acid position, the number of the amino acid position is counted from the amino-terminus of a given polypeptide. The first amino acid residue at the amino-terminus of a polypeptide is denoted as position 1. In some embodiments, the first amino acid residue is a Methionine. The position numbers then increase numerically with each amino acid residue from the amino terminus of the polypeptide to the carboxy terminus of the polypeptide, with the last amino acid position being the last amino acid residue at the carboxy terminus of the polypeptide.

[0081] An RGN polypeptide of the disclosure can comprise an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1, and wherein the amino acid residue(s) at one or more of amino acid positions 517, 530, 538, 552, 677, and 801 of said RGN polypeptide differs from the corresponding amino acid residue of SEQ ID NO: 1. In some embodiments, the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1, and wherein the amino acid residue(s) at one or more of amino acid positions 517, 530, 538, 552, 677, and 801 of said RGN polypeptide is a positively charged amino acid residue. In some embodiments, the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1, and wherein the amino acid residue(s) at one or more of amino acid positions 517, 530, 538, 552, 677, and 801 of said RGN polypeptide is an R. In some embodiments, the RGN polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1, and wherein the amino acid residue(s) at one or more of amino acid positions 517, 530, 538, 552, 677, and 801 of said RGN polypeptide is an R. An RGN polypeptide of the disclosure can comprise the amino acid sequence set forth as any one of SEQ ID NOs: 450, 463, 471, 485, 602, and 717.

[0082] An RGN polypeptide of the disclosure can comprise an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1, and wherein the amino acid residues are arginines (Rs) at a combination of amino acid positions selected from the following combinations of amino acid positions of said RGN polypeptide: (a) amino acid positions 517, 530, 552, and 677; (b) amino acid positions 530, 538, 552, and 801; (c) amino acid positions 530, 552, 677, and 801; (d) amino acid positions 530, 577, and 790; (e) amino acid positions 517, 530, 538, 552, and 677; (f) amino acid positions 530, 538, 677, and 801; (g) amino acid positions 538, 552, 677, and 801; (h) amino acid positions 517, 530, 538, and 552; (i) amino acid positions 517 and 530; (j) amino acid positions 530, 538, and 552; (k) amino acid positions 517, 530, 538, and 677; (1) amino acid positions 517, 552, 677, and 801; (m) amino acid positions 530, 677, and 801; (n) amino acid positions 517, 530, 677, and 801; (o) amino acid positions 517, 530, and 677; (p) amino acid positions 530, 538, 552, and 677; (q) amino acid positions 517, 552, and 677; and (r) amino acid positions 517, 530, 552, 677, and 801. In some embodiments, the RGN polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1, and wherein the amino acid residues are arginines (Rs) at a combination of amino acid positions selected from the following combinations of amino acid positions of said RGN polypeptide: (a) amino acid positions 517, 530, 552, and 677; (b) amino acid positions 530, 538, 552, and 801; (c) amino acid positions 530, 552, 677, and 801; (d) amino acid positions 530, 577, and 790; (e) amino acid positions 517, 530, 538, 552, and 677; (f) amino acid positions 530, 538, 677, and 801; (g) amino acid positions 538, 552, 677, and 801; (h) amino acid positions 517, 530, 538, and 552; (i) amino acid positions 517 and 530; (j) amino acid positions 530, 538, and 552; (k) amino acid positions 517, 530, 538, and 677; (1) amino acid positions 517, 552, 677, and 801; (m) amino acid positions 530, 677, and 801; (n) amino acid positions 517, 530, 677, and 801; (o) amino acid positions 517, 530, and 677; (p) amino acid positions 530, 538, 552, and 677; (q) amino acid positions 517, 552, and 677; and (r) amino acid positions 517, 530, 552, 677, and 801. An RGN polypeptide of the disclosure can comprise the amino acid sequence set forth as any one of SEQ ID NOs: 1072, 1080, 1082, 1105, 1084, 1081, 1083, 1069, 1034, 1059, 1070, 1078, 1064, 1074, 1051, 1079, 1056, and 1087.

[0083] An RGN polypeptide of the disclosure can comprise an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1, and wherein the amino acid residues are arginines (Rs) at a combination of amino acid positions selected from the following combinations of amino acid positions of said RGN polypeptide: (a) amino acid positions 517, 530, 552, and 677; (b) amino acid positions 530, 538, 552, and 801; and (c) amino acid positions 530, 552, 677, and 801. In some embodiments, the RGN polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1, and wherein the amino acid residues are arginines (Rs) at a combination of amino acid positions selected from the following combinations of amino acid positions of said RGN polypeptide: (a) amino acid positions 517, 530, 552, and 677; (b) amino acid positions 530, 538, 552, and 801; and (c) amino acid positions 530, 552, 677, and 801. An RGN polypeptide of the disclosure can comprise the amino acid sequence set forth as any one of SEQ ID NOs: 1072, 1080, and 1082.

[0084] An RGN polypeptide of the disclosure can comprise an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4 (LPG10241), and wherein the amino acid residue(s) at one or more of amino acid positions 377, 378, 438, 625, 638, 641, 645, 691, 692, 695, 698, 702, 707, 747, and 755 of said RGN polypeptide differs from the corresponding amino acid residue of SEQ ID NO: 4. In some embodiments, the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4, and wherein the amino acid residue(s) at one or more of amino acid positions 377, 378, 438, 625, 638, 641, 645, 691, 692, 695, 698, 702, 707, 747, and 755 of said RGN polypeptide is a positively charged amino acid residue. In some embodiments, the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4, and wherein the amino acid residue(s) at one or more of amino acid positions 377, 378, 438, 625, 638, 641, 645, 691, 692, 695, 698, 702, 707, 747, and 755 of said RGN polypeptide is an R. In some embodiments, the RGN polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4, and wherein the amino acid residue(s) at one or more of amino acid positions 377, 378, 438, 625, 638, 641, 645, 691, 692, 695, 698, 702, 707, 747, and 755 of said RGN polypeptide is an R. An RGN polypeptide of the disclosure can comprise the amino acid sequence set forth as any one of SEQ ID NOs: 809, 810, 866, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 1012, and 1019.

[0085] An RGN polypeptide of the disclosure can comprise an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4, and wherein the amino acid residue(s) at one or more of amino acid positions 377, 625, 638, 691, 698, and 747 of said RGN polypeptide differs from the corresponding amino acid residue of SEQ ID NO: 4. In some embodiments, the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4, and wherein the amino acid residue(s) at one or more of amino acid positions 377, 625, 638, 691, 698, and 747 of said RGN polypeptide is a positively charged amino acid residue. In some embodiments, the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4, and wherein the amino acid residue(s) at one or more of amino acid positions 377, 625, 638, 691, 698, and 747 of said RGN polypeptide is an R. In some embodiments, the RGN polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4, and wherein the amino acid residue(s) at one or more of amino acid positions 377, 625, 638, 691, 698, and 747 of said RGN polypeptide is an R. An RGN polypeptide of the disclosure can comprise the amino acid sequence set forth as any one of SEQ ID NOs: 809, 898, 909, 960, 966, and 1012.

[0086] An RGN polypeptide of the disclosure can comprise an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4, and wherein the amino acid residues are arginines (Rs) at a combination of amino acid positions selected from the following combinations of amino acid positions of said RGN polypeptide: (a) amino acid positions 377, 625, and 698; (b) amino acid positions 377, 625, 638, 698, and 747; (c) amino acid positions 377, 625, 691, 698, and 747; (d) amino acid positions 377, 638, 691, and 747; (e) amino acid positions 377, 638, 691, 698, and 747; (f) amino acid positions 377, 625, and 691; (g) amino acid positions 377, 625, 698, and 747; (h) amino acid positions 377, 638, 691, and 698; (i) amino acid positions 377, 625, and 638; (j) amino acid positions 377, 638, 698, and 747; (k) amino acid positions 377, 625, 638, 691, and 698; (1) amino acid positions 625, 638, and 698; (m) amino acid positions 377, 625, 691, and 698; (n) amino acid positions 377, 638, and 698; (o) amino acid positions 625, 691, 698, and 747; (p) amino acid positions 638 and 698; and (q) amino acid positions 377 and 691. In some embodiments, the RGN polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4, and wherein the amino acid residues are arginines (Rs) at a combination of amino acid positions selected from the following combinations of amino acid positions of said RGN polypeptide: (a) amino acid positions 377, 625, and 698; (b) amino acid positions 377, 625, 638, 698, and 747; (c) amino acid positions 377, 625, 691, 698, and 747; (d) amino acid positions 377, 638, 691, and 747; (e) amino acid positions 377, 638, 691, 698, and 747; (f) amino acid positions 377, 625, and 691; (g) amino acid positions 377, 625, 698, and 747; (h) amino acid positions 377, 638, 691, and 698; (i) amino acid positions 377, 625, and 638; (j) amino acid positions 377, 638, 698, and 747; (k) amino acid positions 377, 625, 638, 691, and 698; (1) amino acid positions 625, 638, and 698; (m) amino acid positions 377, 625, 691, and 698; (n) amino acid positions 377, 638, and 698; (o) amino acid positions 625, 691, 698, and 747; (p) amino acid positions 638 and 698; and (q) amino acid positions 377 and 691. An RGN polypeptide of the disclosure can comprise the amino acid sequence set forth as any one of SEQ ID NOs: 1123, 1158, 1159, 1148, 1160, 1122, 1146, 1147, 1121, 1149, 1156, 1132, 1144, 1126, 1154, 1116, and 1108.

[0087] An RGN polypeptide of the disclosure can comprise an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4, and wherein the amino acid residues are arginines (Rs) at a combination of amino acid positions selected from the following combinations of amino acid positions of said RGN polypeptide: (a) amino acid positions 377, 625, and 698; (b) amino acid positions 377, 625, 638, 698, and 747; and (c) amino acid positions 377, 625, 691, 698, and 747. In some embodiments, the RGN polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4, and wherein the amino acid residues are arginines (Rs) at a combination of amino acid positions selected from the following combinations of amino acid positions of said RGN polypeptide: (a) amino acid positions 377, 625, and 698; (b) amino acid positions 377, 625, 638, 698, and 747; and (c) amino acid positions 377, 625, 691, 698, and 747. An RGN polypeptide of the disclosure can comprise the amino acid sequence set forth as any one of SEQ ID NOs: 1123, 1158, and 1159.

[0088] An RGN polypeptide of the disclosure can comprise an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4, and wherein the amino acid residue(s) at one or more of amino acid positions 685, 691, 692, 695, 698, 702, 707, 736, 740, 743, 747, 751, 752, 753, 755, 757, 758, 760, 764, and 766 of said RGN polypeptide differs from the corresponding amino acid residue of SEQ ID NO: 4. In some embodiments, the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4, and wherein the amino acid residue(s) at one or more of amino acid positions 685, 691, 692, 695, 698, 702, 707, 736, 740, 743, 747, 751, 752, 753, 755, 757, 758, 760, 764, and 766 of said RGN polypeptide is a positively charged amino acid residue. In some embodiments, the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4, and wherein the amino acid residue(s) at one or more of amino acid positions 685, 691, 692, 695, 698, 702, 707, 736, 740, 743, 747, 751, 752, 753, 755, 757, 758, 760, 764, and 766 of said RGN polypeptide is an R or a lysine (K). In some embodiments, the RGN polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4, and wherein the amino acid residue(s) at one or more of amino acid positions 685, 691, 692, 695, 698, 702, 707, 736, 740, 743, 747, 751, 752, 753, 755, 757, 758, 760, 764, and 766 of said RGN polypeptide is an R or K.

[0089] An RGN polypeptide of the disclosure can comprise an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4, and wherein the amino acid residues are arginines (Rs) and / or lysines (Ks) at a combination of amino acid positions selected from the following combinations of amino acid positions of said RGN polypeptide: (a) amino acid positions 736, 743, 752, 753, 755, 757, 758, and 760; (b) amino acid positions 740, 747, 751, 753, 755, 760, 764, and 766; (c) amino acid positions 685, 691, and 698; and (d) amino acid positions 685, 692, 695, 702, and 707. An RGN polypeptide of the disclosure can comprise the amino acid sequence set forth as any one of SEQ ID NOs: 1168, 1171, 1169, and 1170.

[0090] An RGN polypeptide of the disclosure can comprise an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4, and wherein said RGN polypeptide comprises a deletion of amino acid residues corresponding to amino acid residues 751-769 of SEQ ID NO: 4. In some embodiments, an RGN polypeptide of the disclosure can comprise the amino acid sequence set forth as SEQ ID NO: 1173.

[0091] An RGN polypeptide of the disclosure can comprise an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1, and wherein one or more of amino acid positions 293, 294, 359, 360, 361, 401, 402, 403, 596, and 862 of said RGN polypeptide comprises substitution or insertion of one or more glycines (G) immediately N-terminal to, at, or immediately C- terminal to said amino acid position. In some embodiments, the RGN polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1, and wherein one or more of amino acid positions 293, 294, 359, 360, 361, 401, 402, 403, 596, and 862 of said RGN polypeptide comprises substitution or insertion of one or more G immediately N-terminal to, at, or immediately C-terminal to said amino acid position. In some embodiments, the substitution or insertion of one or more G comprises substitution or insertion of one, two, or three G. In some embodiments, the substitution or insertion of one or more G is selected from: (a) an insertion of three G between amino acid positions 293 and 294; (b) an insertion of two G between amino acid positions 359 and 360; (c) a substitution of G at amino acid position 361; (d) an insertion of one G between amino acid positions 402 and 403; (e) a substitution of G at amino acid position 401; (f) a substitution of G at amino acid position 596; and (g) a substitution of G at amino acid position 862. An RGN polypeptide of the disclosure can comprise the amino acid sequence set forth as any one of SEQ ID NOs: 1304, 1322, 1324, 1328, 1330, 1361, and 1400.

[0092] The terms ‘immediately N-terminal to’ and ‘immediately C-terminal to’, in the context of substitution or insertion of one or more G in an RGN polypeptide, refers to substitution or insertion at the first amino acid position N-terminal to, or the first amino acid position C-terminal to, respectively, the recited amino acid position.

[0093] An RGN polypeptide of the disclosure can comprise an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4, and wherein one or more of amino acid positions 566, 567, and 584 of said RGN polypeptide comprises substitution or insertion of one or more glycines (G) immediately N-terminal to, at, or immediately C-terminal to said amino acid position. In some embodiments, the RGN polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4, and wherein one or more of amino acid positions 566, 567, and 584 of said RGN polypeptide comprises substitution or insertion of one or more G immediately N-terminal to, at, or immediately C- terminal to said amino acid position. In some embodiments, the substitution or insertion of one or more G comprises substitution or insertion of one, two, or three G. In some embodiments, the substitution or insertion of one or more G is selected from: (a) a substitution of G at amino acid position 566 and a substitution of G at amino acid position 567; and (b) a substitution of G at amino acid position 584. An RGN polypeptide of the disclosure can comprise the amino acid sequence selected from: (a) the amino acid sequence set forth as SEQ ID NO: 1223; and (b) the amino acid sequence set forth as SEQ ID NO: 1228.

[0094] An RGN polypeptide of the disclosure can comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a variant LPG10238 RGN polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 1087, 1105, 1304, 1322, 1324, 1328, 1330, 1361, 1400, and 2686, wherein the RGN polypeptide has RNA-guided sequence-specific binding activity. In some embodiments, an RGN polypeptide of the disclosure comprises the amino acid sequence set forth as any one of SEQ ID NOs: 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 1087, 1105, 1304, 1322, 1324, 1328, 1330, 1361, 1400, and 2686.

[0095] An RGN polypeptide of the disclosure can comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a variant LPG10241 RGN polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1108, 1116, 1121, 1122, 1123, 1126, 1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173, 1223, 1228, and 2687, wherein the RGN polypeptide has RNA-guided sequence-specific binding activity. In some embodiments, an RGN polypeptide of the disclosure comprises the amino acid sequence set forth as any one of SEQ ID NOs: 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1108, 1116, 1121, 1122, 1123, 1126, 1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173, 1223, 1228, and 2687.

[0096] An RGN of the disclosure binds to a target sequence, e.g., such as those that are disclosed herein. In some embodiments, an RGN, or an active variant or fragment thereof, of the disclosure recognizes a protospacer adjacent motif (PAM) having a consensus nucleotide sequence including a AYG 5' of the target sequence on its non-target strand (where Y is C or T / U). In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1 (LPG10238), or an active variant or fragment thereof, recognizes a PAM having a consensus nucleotide sequence as illustrated in FIG. 3 A 5' of the target sequence on its nontarget strand.

[0097] In some embodiments, an RGN, or an active variant or fragment thereof, of the disclosure recognizes a PAM having a consensus nucleotide sequence including a ATTN 5' of the target sequence on its non-target strand (where N is A, C, T / U, or G). In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 2 (LPG10239), or an active variant or fragment thereof, recognizes a PAM having a consensus nucleotide sequence as illustrated in FIG. 3B 5' of the target sequence on its non- target strand.

[0098] In some embodiments, an RGN, or an active variant or fragment thereof, of the disclosure recognizes a PAM having a consensus nucleotide sequence including a VTTN 5' of the target sequence on its non-target strand (where N is A, C, T / U, or G; and V is A or G or C). In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 3 (LPG10240), or an active variant or fragment thereof, recognizes a PAM having a consensus nucleotide sequence as illustrated in FIG. 3C 5' of the target sequence on its non-target strand.

[0099] In some embodiments, an RGN, or an active variant or fragment thereof, of the disclosure recognizes a PAM having a consensus nucleotide sequence including a TTN 5' of the target sequence on its non-target strand (where N is A, C, T / U, or G). In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 4 (LPG10241), or an active variant or fragment thereof, recognizes a PAM having a consensus nucleotide sequence as illustrated in FIG. 3D 5' of the target sequence on its nontarget strand.

[0100] In some embodiments, an RGN, or an active variant or fragment thereof, of the disclosure recognizes a PAM having a consensus nucleotide sequence including a TTN 5' of the target sequence on its non-target strand (where N is A, C, T / U, or G). In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1930 (LPG13090), or an active variant or fragment thereof, recognizes a PAM having a consensus nucleotide sequence as illustrated in FIG. 3E 5’ of the target sequence on its nontarget strand.

[0101] In some embodiments, an RGN, or an active variant or fragment thereof, of the disclosure recognizes a PAM having a consensus nucleotide sequence including a STTN 5' of the target sequence on its non-target strand (where S is C or G; and N is A, C, T / U, or G). In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1931 (LPG13091), or an active variant or fragment thereof, recognizes a PAM having a consensus nucleotide sequence as illustrated in FIG. 3F 5' of the target sequence on its non-target strand.

[0102] In some embodiments, an RGN, or an active variant or fragment thereof, of the disclosure recognizes a PAM having a consensus nucleotide sequence including a TTN 5' of the target sequence on its non-target strand (where N is A, C, T / U, or G). In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1932 (LPG13092), or an active variant or fragment thereof, recognizes a PAM having a consensus nucleotide sequence as illustrated in FIG. 3G 5' of the target sequence on its non- target strand.

[0103] In some embodiments, an RGN, or an active variant or fragment thereof, of the disclosure recognizes a PAM having a consensus nucleotide sequence including a ATTN 5' of the target sequence on its non-target strand (where N is A, C, T / U, or G). In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1933 (LPG13093), or an active variant or fragment thereof, recognizes a PAM having a consensus nucleotide sequence as illustrated in FIG. 3H 5' of the target sequence on its non- target strand.

[0104] In some embodiments, an RGN, or an active variant or fragment thereof, of the disclosure recognizes a PAM having a consensus nucleotide sequence including a TTN 5' of the target sequence on its non-target strand (where N is A, C, T / U, or G). In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1934 (LPG13094), or an active variant or fragment thereof, recognizes a PAM having a consensus nucleotide sequence as illustrated in FIG. 31 5' of the target sequence on its non- target strand.

[0105] In some embodiments, an RGN, or an active variant or fragment thereof, of the disclosure recognizes a PAM having a consensus nucleotide sequence including a TTH 5' of the target sequence on its non-target strand (where H is A or C or T / U). In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1935 (LPG13095), or an active variant or fragment thereof, recognizes a PAM having a consensus nucleotide sequence as illustrated in FIG. 3J 5' of the target sequence on its nontarget strand.

[0106] In some embodiments, an RGN, or an active variant or fragment thereof, of the disclosure recognizes a PAM having a consensus nucleotide sequence including a TTN 5' of the target sequence on its non-target strand (where N is A, C, T / U, or G). In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1936 (LPG13096), or an active variant or fragment thereof, recognizes a PAM having a consensus nucleotide sequence as illustrated in FIG. 3K 5' of the target sequence on its nontarget strand.

[0107] In some embodiments, an RGN, or an active variant or fragment thereof, of the disclosure recognizes a PAM having a consensus nucleotide sequence including a RTTN 5' of the target sequence on its non-target strand (where R is A or G; and N is A, C, T / U, or G). In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1937 (LPG13097), or an active variant or fragment thereof, recognizes a PAM having a consensus nucleotide sequence as illustrated in FIG. 3L 5' of the target sequence on its non-target strand.

[0108] In some embodiments, an RGN, or an active variant or fragment thereof, of the disclosure recognizes a PAM having a consensus nucleotide sequence including a VTTN 5' of the target sequence on its non-target strand (where V is A or G or C; and N is A, C, T / U, or G). In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1938 (LPG13098), or an active variant or fragment thereof, recognizes a PAM having a consensus nucleotide sequence as illustrated in FIG. 3M 5' of the target sequence on its non-target strand.

[0109] In some embodiments, an RGN, or an active variant or fragment thereof, of the disclosure recognizes a PAM having a consensus nucleotide sequence including a TTN 5' of the target sequence on its non-target strand (where N is A, C, T / U, or G). In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1939 (LPG13099), or an active variant or fragment thereof, recognizes a PAM having a consensus nucleotide sequence as illustrated in FIG. 3N 5' of the target sequence on its non- target strand.

[0110] In some embodiments, an RGN, or an active variant or fragment thereof, of the disclosure recognizes a PAM having a consensus nucleotide sequence including a TTN 5' of the target sequence on its non-target strand (where N is A, C, T / U, or G). In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1940 (LPG13100), or an active variant or fragment thereof, recognizes a PAM having a consensus nucleotide sequence as illustrated in FIG. 30 5' of the target sequence on its non- target strand.

[0111] In some embodiments, an RGN, or an active variant or fragment thereof, of the disclosure recognizes a PAM having a consensus nucleotide sequence including a TTN 5' of the target sequence on its non-target strand (where N is A, C, T / U, or G). In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1941 (LPG13101), or an active variant or fragment thereof, recognizes a PAM having a consensus nucleotide sequence as illustrated in FIG. 3P 5' of the target sequence on its nontarget strand.

[0112] In some embodiments, an RGN, or an active variant or fragment thereof, of the disclosure recognizes a PAM having a consensus nucleotide sequence including a TTN 5' of the target sequence on its non-target strand (where N is A, C, T / U, or G). In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1942 (LPG13102), or an active variant or fragment thereof, recognizes a PAM having a consensus nucleotide sequence as illustrated in FIG. 3Q 5' of the target sequence on its nontarget strand.

[0113] In some embodiments, an RGN, or an active variant or fragment thereof, of the disclosure recognizes a PAM having a consensus nucleotide sequence including a VTTN 5' of the target sequence on its non-target strand (where V is A or G or C; and N is A, C, T / U, or G). In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1943 (LPG13103), or an active variant or fragment thereof, recognizes a PAM having a consensus nucleotide sequence as illustrated in FIG. 3R 5' of the target sequence on its non-target strand.

[0114] In some embodiments, an RGN, or an active variant or fragment thereof, of the disclosure recognizes a PAM having a consensus nucleotide sequence including a ATTN 5' of the target sequence on its non-target strand (where N is A, C, T / U, or G). In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1944 (LPG13104), or an active variant or fragment thereof, recognizes a PAM having a consensus nucleotide sequence as illustrated in FIG. 3S 5' of the target sequence on its non- target strand.

[0115] In some embodiments, an RGN, or an active variant or fragment thereof, of the disclosure recognizes a PAM having a consensus nucleotide sequence including a VTTN 5' of the target sequence on its non-target strand (where V is A or G or C; and N is A, C, T / U, or G). In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1945 (LPG13105), or an active variant or fragment thereof, recognizes a PAM having a consensus nucleotide sequence as illustrated in FIG. 3T 5' of the target sequence on its non-target strand.

[0116] In some embodiments, an RGN, or an active variant or fragment thereof, of the disclosure recognizes a PAM having a consensus nucleotide sequence including a TTN 5' of the target sequence on its non-target strand (where N is A, C, T / U, or G). In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1946 (LPG13106), or an active variant or fragment thereof, recognizes a PAM having a consensus nucleotide sequence as illustrated in FIG. 3U 5' of the target sequence on its non- target strand.

[0117] In some embodiments, an RGN, or an active variant or fragment thereof, of the disclosure recognizes a PAM having a consensus nucleotide sequence including a VTTN 5' of the target sequence on its non-target strand (where V is A or G or C; and N is A, C, T / U, or G). In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1947 (LPG13107), or an active variant or fragment thereof, recognizes a PAM having a consensus nucleotide sequence as illustrated in FIG. 3V 5' of the target sequence on its non-target strand.

[0118] In some embodiments, an RGN, or an active variant or fragment thereof, of the disclosure recognizes a PAM having a consensus nucleotide sequence including a TTN 5' of the target sequence on its non-target strand (where N is A, C, T / U, or G). In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1948 (LPG13108), or an active variant or fragment thereof, recognizes a PAM having a consensus nucleotide sequence as illustrated in FIG. 3W 5' of the target sequence on its non- target strand.

[0119] In some embodiments, an RGN, or an active variant or fragment thereof, of the disclosure recognizes a PAM having a consensus nucleotide sequence including a VTTN 5' of the target sequence on its non-target strand (where V is A or G or C; and N is A, C, T / U, or G). In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1949 (LPG13109), or an active variant or fragment thereof, recognizes a PAM having a consensus nucleotide sequence as illustrated in FIG. 3X 5' of the target sequence on its non-target strand.

[0120] In some embodiments, an RGN, or an active variant or fragment thereof, of the disclosure recognizes a PAM having a consensus nucleotide sequence including a RTTN 5' of the target sequence on its non-target strand (where R is A or G; and N is A, C, T / U, or G). In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1950 (LPG13110), or an active variant or fragment thereof, recognizes a PAM having a consensus nucleotide sequence as illustrated in FIG. 3Y 5' of the target sequence on its non-target strand.

[0121] In some embodiments, an RGN, or an active variant or fragment thereof, of the disclosure recognizes a PAM having a consensus nucleotide sequence including a RTYN 5' of the target sequence on its non-target strand (where R is A or G; Y is C or T / U; and N is A, C, T / U, or G). In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1951 (LPG13111), or an active variant or fragment thereof, recognizes a PAM having a consensus nucleotide sequence as illustrated in FIG. 3Z 5' of the target sequence on its non-target strand.

[0122] In some embodiments, an RGN, or an active variant or fragment thereof, of the disclosure recognizes a PAM having a consensus nucleotide sequence including a TTN 5' of the target sequence on its non-target strand (where N is A, C, T / U, or G). In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1952 (LPG13112), or an active variant or fragment thereof, recognizes a PAM having a consensus nucleotide sequence as illustrated in FIG. 3AA 5' of the target sequence on its non- target strand.

[0123] In some embodiments, an RGN, or an active variant or fragment thereof, of the disclosure recognizes a PAM having a consensus nucleotide sequence including a ATG 5' of the target sequence on its non-target strand. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1953 (LPG13113), or an active variant or fragment thereof, recognizes a PAM having a consensus nucleotide sequence as illustrated in FIG. 3BB 5' of the target sequence on its non-target strand. In some embodiments, an RGN, or an active variant or fragment thereof, of the disclosure recognizes a PAM having a consensus nucleotide sequence including a TTN 5' of the target sequence on its non-target strand (where N is A, C, T / U, or G). In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1954 (LPG13114), or an active variant or fragment thereof, recognizes a PAM having a consensus nucleotide sequence as illustrated in FIG. 3CC 5' of the target sequence on its nontarget strand.

[0124] In some embodiments, an RGN, or an active variant or fragment thereof, of the disclosure recognizes a PAM having a consensus nucleotide sequence including a TTN 5' of the target sequence on its non-target strand (where N is A, C, T / U, or G). In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1955 (LPG13115), or an active variant or fragment thereof, recognizes a PAM having a consensus nucleotide sequence as illustrated in FIG. 3DD 5' of the target sequence on its non- target strand.

[0125] In some embodiments, an RGN, or an active variant or fragment thereof, of the disclosure recognizes a PAM having a consensus nucleotide sequence including a TTN 5' of the target sequence on its non-target strand (where N is A, C, T / U, or G). In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1956 (LPG13116), or an active variant or fragment thereof, recognizes a PAM having a consensus nucleotide sequence as illustrated in FIG. 3EE 5' of the target sequence on its non- target strand.

[0126] As used herein in reference to a PAM recognition nucleotide motif, Y refers to cytosine (C) or thymine / uracil (T / U); N refers to adenine (A) or cytosine (C) or thymine / uracil (T / U) or guanine (G); V refers to adenine (A) or cytosine (C) or guanine (G); S refers to cytosine (C) or guanine (G); H refers to adenine (A) or cytosine (C) or thymine / uracil (T / U); and R refers to adenine (A) or guanine (G). In some embodiments, an active fragment or variant of an RGN recognizing such PAM sequences is capable of binding and in some embodiments, cleaving or nicking a target sequence.

[0127] RGNs provided herein can comprise at least one nuclease domain (e.g., DNase, RNase domain) and at least one RNA recognition and / or RNA binding domain to interact with guide RNAs. In some embodiments, Type V RGNs of the disclosure comprise only one nuclease domain. In some embodiments, the Type V RGN nuclease domain is a RuvC domain. In some embodiments, Type V RGNs of the disclosure do not comprise an HNH nuclease domain. Further domains that can be found in RGNs provided herein include, but are not limited to: DNA binding domains, helicase domains, protein-protein interaction domains, and dimerization domains. In some embodiments, the RGNs provided herein can comprise at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to one or more of a DNA binding domain, helicase domain, protein-protein interaction domain, and dimerization domain of a Type V RGN.

[0128] An RGN polypeptide of the disclosure can comprise one or more of the following domains: a PAM- interacting (PI) domain, a Rec domain (Reel, RecII), an oligo-binding domain (OBD), a zinc-binding domain (e.g., comprising a Zinc finger), or a RuvC nuclease domain. The RuvC domain can include a RuvCI, a RuvCII, a RuvCIII domain, or a combination thereof. A Rec or recognition lobe mediates nucleic acid binding through multiple Rec domains (e.g., RecI-III). The general domains of RGN polypeptides can be determined via structural comparison to RGN polypeptides with defined domains.

[0129] Non-limiting examples of domains within LPG10241 RGN (SEQ ID NO: 4) or a variant LPG10241 RGN (SEQ ID NOs: 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1108, 1116, 1121, 1122, 1123, 1126, 1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173, 1223, 1228, and 2687) include: PI from amino acid residues 1 to 59; Reel from amino acid residues 74 to 216; OBD from amino acid residues 216 to 370; RuvC-I from amino acid residues 370 to 445; RecII from amino acid residues 445 to 594; RuvC-II from amino acid residues 594 to 681; zinc-binding domain (ZF) from amino acid residues 681 to 710; and RuvC-III from amino acid residues 710 to 770. The LPG10241 mapped domain organization is shown in FIG. 4A.

[0130] Non-limiting examples of domains within LPG10238 RGN (SEQ ID NO: 1) or a variant LPG10238 RGN (SEQ ID NOs: 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 1087, 1105, 1304, 1322, 1324, 1328, 1330, 1361, 1400, and 2686) include: Red from amino acid residues 66 to 160; RuvC-I from amino acid residues 473 to 527; Redl from amino acid residues 527 to 667; RuvC-II from amino acid residues 667 to 725; and RuvC-III from amino acid residues 732 to 889. The LPG10238 mapped domain organization is shown in FIG. 4B.

[0131] An RGN, or an active variant or fragment thereof, of the disclosure can have a PAM-interacting (PI) domain that contributes to recognition of a PAM site in a target nucleic acid molecule. The PI domain can comprise 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, or more amino acid residues. In some embodiments, the PI domain of an RGN, or an active variant or fragment thereof, of the disclosure is located within the amino (N)-terminal region of the RGN. The N-terminal region comprising the PI domain of an RGN, or an active variant or fragment thereof, of the disclosure can include the N- terminal 100 amino acid residues (i.e. amino acid residues 1 to 100 starting from the N-terminus of the RGN), the N-terminal 90 amino acid residues, the N-terminal 80 amino acid residues, the N-terminal 70 amino acid residues, the N-terminal 60 amino acid residues, the N-terminal 50 amino acid residues, the N- terminal 40 amino acid residues, the N-terminal 30 amino acid residues, the N-terminal 20 amino acid residues, or the N-terminal 10 amino acid residues of the RGN. In some embodiments, the PI domain of an RGN, or an active variant or fragment thereof, of the disclosure is within or includes amino acid residues 1 to 59 (i.e., amino acid residues 1 to 59 starting from the N-terminus) of the RGN. In some embodiments, the PI domain of an RGN having the amino acid sequence set forth as SEQ ID NO: 2, or an active variant or fragment thereof, is within or includes amino acid residues 1 to 59 of the RGN. In some embodiments, the PI domain of an RGN having the amino acid sequence set forth as SEQ ID NO: 3, or an active variant or fragment thereof, is within or includes amino acid residues 1 to 59 of the RGN. In some embodiments, the PI domain of an RGN having the amino acid sequence set forth as SEQ ID NO: 4, or an active variant or fragment thereof, is within or includes amino acid residues 1 to 59 of the RGN. An active variant or fragment of a variant LPG10238 RGN disclosed herein can comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to amino acid residues 1 to 472 of any one of SEQ ID NOs: 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 1087, 1105, 1304, 1322, 1324, 1328, 1330, 1361, 1400, and 2686, and can comprise amino acid residues having 100% sequence identity to amino acid residues 473 to 889 of the corresponding variant LPG10238 RGN or that differs from amino acid residues 473 to 889 by 1, 2, 3, or 4 amino acid residues.

[0132] An active variant or fragment of a variant LPG 10241 RGN disclosed herein can comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to amino acid residues 1 to 369 of any one of SEQ ID NOs: 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1108, 1116, 1121, 1122, 1123, 1126, 1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173, 1223, 1228, and 2687, and can comprise amino acid residues having 100% sequence identity to amino acid residues 370 to 770 of the corresponding variant LPG10241 RGN or that differs from amino acid residues 370 to 770 by 1, 2, 3, or 4 amino acid residues.

[0133] In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as AYG. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO:

[0134] 1, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as AYG, when bound to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) sequence set forth as SEQ ID NO: 5 or 1631, or an active variant or fragment thereof.

[0135] In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 2, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as ATTN. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO:

[0136] 2, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as ATTN, when bound to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) sequence set forth as SEQ ID NO: 6, or an active variant or fragment thereof.

[0137] In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 3, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as VTTN. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO:

[0138] 3, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as VTTN, when bound to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) sequence set forth as SEQ ID NO: 7, or an active variant or fragment thereof. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 4, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTN. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 4, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTN, when bound to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) sequence set forth as any one of SEQ ID NOs: 8, and 1617-1620, or an active variant or fragment thereof.

[0139] In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1930, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTN. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1930, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTN, when bound to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) sequence set forth as SEQ ID NO: 1957, or an active variant or fragment thereof.

[0140] In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1931, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as STTN. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1931, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as STTN, when bound to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) sequence set forth as SEQ ID NO: 1958, or an active variant or fragment thereof.

[0141] In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1932, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTN. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1932, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTN, when bound to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) sequence set forth as SEQ ID NO: 1959, or an active variant or fragment thereof.

[0142] In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1933, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as ATTN. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1933, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as ATTN, when bound to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) sequence set forth as SEQ ID NO: 1960, or an active variant or fragment thereof.

[0143] In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1934, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTN. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1934, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTN, when bound to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) sequence set forth as SEQ ID NO: 1961, or an active variant or fragment thereof. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1935, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTH. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1935, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTH, when bound to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) sequence set forth as SEQ ID NO: 1962, or an active variant or fragment thereof.

[0144] In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1936, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTN. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1936, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTN, when bound to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) sequence set forth as SEQ ID NO: 1963, or an active variant or fragment thereof.

[0145] In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1937, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as RTTN. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1937, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as RTTN, when bound to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) sequence set forth as SEQ ID NO: 1964, or an active variant or fragment thereof.

[0146] In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1938, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as VTTN. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1938, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as VTTN, when bound to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) sequence set forth as SEQ ID NO: 1965, or an active variant or fragment thereof.

[0147] In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1939, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTN. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1939, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTN, when bound to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) sequence set forth as SEQ ID NO: 1966, or an active variant or fragment thereof.

[0148] In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1940, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTN. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1940, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTN, when bound to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) sequence set forth as SEQ ID NO: 1967, or an active variant or fragment thereof. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1941, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTN. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1941, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTN, when bound to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) sequence set forth as SEQ ID NO: 1968, or an active variant or fragment thereof.

[0149] In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1942, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTN. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1942, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTN, when bound to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) sequence set forth as SEQ ID NO: 1969, or an active variant or fragment thereof.

[0150] In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1943, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as VTTN. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1943, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as VTTN, when bound to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) sequence set forth as SEQ ID NO: 1970, or an active variant or fragment thereof.

[0151] In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1944, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as ATTN. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1944, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as ATTN, when bound to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) sequence set forth as SEQ ID NO: 1971, or an active variant or fragment thereof.

[0152] In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1945, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as VTTN. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1945, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as VTTN, when bound to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) sequence set forth as SEQ ID NO: 1972, or an active variant or fragment thereof.

[0153] In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1946, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTN. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1946, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTN, when bound to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) sequence set forth as SEQ ID NO: 1973, or an active variant or fragment thereof. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1947, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as VTTN. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1947, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as VTTN, when bound to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) sequence set forth as SEQ ID NO: 1974, or an active variant or fragment thereof.

[0154] In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1948, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTN. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1948, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTN, when bound to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) sequence set forth as SEQ ID NO: 1975, or an active variant or fragment thereof.

[0155] In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1949, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as VTTN. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1949, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as VTTN, when bound to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) sequence set forth as SEQ ID NO: 1976, or an active variant or fragment thereof.

[0156] In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1950, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as RTTN. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1950, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as RTTN, when bound to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) sequence set forth as SEQ ID NO: 1977, or an active variant or fragment thereof.

[0157] In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1951, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as RTYN. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1951, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as RTYN, when bound to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) sequence set forth as SEQ ID NO: 1978, or an active variant or fragment thereof.

[0158] In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1952, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTN. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1952, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTN, when bound to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) sequence set forth as SEQ ID NO: 1979, or an active variant or fragment thereof. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1953, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as ATG. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1953, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as ATG, when bound to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) sequence set forth as SEQ ID NO: 1980, or an active variant or fragment thereof.

[0159] In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1954, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTN. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1954, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTN, when bound to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) sequence set forth as SEQ ID NO: 1981, or an active variant or fragment thereof.

[0160] In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1955, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTN. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1955, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTN, when bound to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) sequence set forth as SEQ ID NO: 1982, or an active variant or fragment thereof.

[0161] In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1956, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTN. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1956, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTN, when bound to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) sequence set forth as SEQ ID NO: 1983, or an active variant or fragment thereof.

[0162] In various embodiments, a target sequence is bound by an RGN provided herein. In those instances wherein the target sequence is double -stranded (e.g., double -stranded DNA), the target strand of the target sequence hybridizes with the guide RNA associated with the RGN. The target strand and / or the non-target strand of the target sequence (e.g., target DNA sequence) can then be subsequently cleaved by the RGN if the polypeptide possesses nuclease activity. The terms “cleave” or “cleavage” refer to the hydrolysis of at least one phosphodiester bond within the backbone of one or both strands of a double-stranded target sequence (e.g., target DNA sequence) that can result in either single-stranded or double -stranded breaks within the target sequence. The presently disclosed RGNs can cleave nucleotides within a polynucleotide, functioning as an endonuclease or can be an exonuclease, removing successive nucleotides from the end (the 5' and / or the 3' end) of a polynucleotide. In some embodiments, the disclosed RGNs can cleave nucleotides of a target polynucleotide within any position of a polynucleotide and thus function as both an endonuclease and exonuclease. The cleavage of a target polynucleotide by the presently disclosed RGNs can result in staggered breaks or blunt ends. A blunt cut generates two blunt ends, such that each blunt end of the cut nucleic acid molecule has both strands that are of equal length - i.e. there are no unpaired bases on either strand of a blunt end.

[0163] In some embodiments, Type V RGNs of the disclosure generate staggered cuts in a nucleic acid molecule. A staggered cut in a nucleic acid molecule leads to two sticky ends or overhanging ends, and is formed when the nuclease cuts each strand of a nucleic acid molecule such that the cuts are not directly opposite each other. For each sticky end of the cut nucleic acid molecule, one strand (i.e. the overhanging strand) is longer than the other (typically by at least a few nucleotides), such that the longer strand has bases which are left unpaired. The longer strand of an overhanging end of a cleaved nucleic acid molecule can have one unpaired nucleotide, two unpaired nucleotides, 3 unpaired nucleotides, 4 unpaired nucleotides, 5 unpaired nucleotides, 6 unpaired nucleotides, 7 unpaired nucleotides, 8 unpaired nucleotides, 9 unpaired nucleotides, 10 unpaired nucleotides, 11 unpaired nucleotides, 12 unpaired nucleotides, or more unpaired nucleotides. In some embodiments, the longer strand of an overhanging end of a cleaved nucleic acid molecule can have one unpaired nucleotide. The overhanging end of a cleaved nucleic acid molecule can be a 3' overhang or a 5' overhang. In some embodiments, the overhanging end of a cleaved nucleic acid molecule is a 3' overhang. In some embodiments, the overhanging end of a cleaved nucleic acid molecule is a 5' overhang. In some embodiments, an RGN, or an active variant or fragment thereof, of the disclosure cleaves a target nucleic acid molecule to form a staggered cut, wherein the staggered cut creates a 3' overhang with one unpaired nucleotide.

[0164] An RGN polypeptide having the amino acid sequence set forth as SEQ ID NO: 1 (LPG10238), or active variants or fragments thereof, can cleave a double-stranded nucleic acid molecule to generate a 3' overhang with one unpaired nucleotide. An RGN polypeptide having the amino acid sequence set forth as SEQ ID NO: 2 (LPG10239), or active variants or fragments thereof, can cleave a double-stranded nucleic acid molecule to generate a 5' overhang with 10 unpaired nucleotides. An RGN polypeptide having the amino acid sequence set forth as SEQ ID NO: 3 (LPG10240), or active variants or fragments thereof, can cleave a double -stranded nucleic acid molecule to generate a 5' overhang with 5 unpaired nucleotides. An RGN polypeptide having the amino acid sequence set forth as SEQ ID NO: 4 (LPG10241), or active variants or fragments thereof, can cleave a double -stranded nucleic acid molecule to generate a 5' overhang with 12 unpaired nucleotides.

[0165] The presently disclosed RGNs can be wild-type sequences derived from bacterial, viral, or archaeal species. Alternatively, the RGNs can be variants or fragments of wild-type polypeptides. The wild-type RGN can be modified to alter nuclease activity or alter PAM specificity, for example. In some embodiments, the RGN is not naturally-occurring.

[0166] In some embodiments, an RGN can function as a nickase, only cleaving a single strand of a doublestranded target sequence (e.g., target DNA sequence). Such RGNs have a single functioning nuclease domain. In particular embodiments, the nickase is capable of cleaving the target strand or the non-target strand of the double -stranded target sequence (e.g., target DNA sequence). In embodiments wherein a nickase is used, in order to effect a double -stranded cleavage of a double -stranded target sequence (e.g., target DNA sequence), two nickases are needed, each of which nicks a single strand within the doublestranded target sequence.

[0167] In other embodiments, the RGN lacks nuclease activity altogether and is referred to herein as catalytically dead, nuclease -dead, or nuclease inactive. In some embodiments, for the Type V RGNs disclosed herein that comprise only one RuvC nuclease domain, catalytically dead, nuclease-dead, or nuclease inactive versions of the enzymes are used for modifying a nucleic acid, such as in base editing as described herein. Any method known in the art for introducing mutations into an amino acid sequence, such as PCR-mediated mutagenesis and site-directed mutagenesis, can be used for generating nickases or nuclease-dead RGNs. See, e.g., U.S. Publ. No. 2014 / 0068797 and U.S. Pat. No. 9,790,490; each of which is incorporated by reference in its entirety. For example, mutating any of the three RuvC catalytic residues in a Type V RGN disclosed herein can generate a catalytically dead enzyme.

[0168] A Type V RGN comprising the amino acid sequence set forth as SEQ ID NO: 1, or an active variant or fragment thereof, can further comprise at least one of the following amino acid mutations to generate a catalytically dead enzyme: D474A, E672A, D759A, wherein the indicated mutated amino acid residues correspond to the amino acid residues of the sequence set forth as SEQ ID NO: 1. A Type V RGN comprising the amino acid sequence set forth as SEQ ID NO: 1, or an active variant or fragment thereof, can further comprise two or three of the following amino acid mutations to generate a catalytically dead enzyme: D474A, E672A, D759A, wherein the indicated mutated amino acid residues correspond to the amino acid residues of the sequence set forth as SEQ ID NO: 1. In some embodiments, a catalytically dead RGN polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence set forth as SEQ ID NO: 2686.

[0169] A Type V RGN comprising the amino acid sequence set forth as SEQ ID NO: 1, or an active variant or fragment thereof, can further comprise at least one of the following amino acid mutations to generate a catalytically dead enzyme: D474R, E672R, D759R, wherein the indicated mutated amino acid residues correspond to the amino acid residues of the sequence set forth as SEQ ID NO: 1 . In some embodiments, a catalytically dead RGN polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence set forth as any one of SEQ ID NOs: 408, 597, and 678. A Type V RGN comprising the amino acid sequence set forth as SEQ ID NO: 1, or an active variant or fragment thereof, can further comprise two or three of the following amino acid mutations to generate a catalytically dead enzyme: D474R, E672R, D759R, wherein the indicated mutated amino acid residues correspond to the amino acid residues of the sequence set forth as SEQ ID NO: 1.

[0170] A Type V RGN comprising the amino acid sequence set forth as SEQ ID NO: 2, or an active variant or fragment thereof, can further comprise at least one of the following amino acid mutations to generate a catalytically dead enzyme: D389A, E600A, D690A, wherein the indicated mutated amino acid residues correspond to the amino acid residues of the sequence set forth as SEQ ID NO: 2. A Type V RGN comprising the amino acid sequence set forth as SEQ ID NO: 2, or an active variant or fragment thereof, can further comprise two or three of the following amino acid mutations to generate a catalytically dead enzyme: D389A, E600A, D690A, wherein the indicated mutated amino acid residues correspond to the amino acid residues of the sequence set forth as SEQ ID NO: 2.

[0171] A Type V RGN comprising the amino acid sequence set forth as SEQ ID NO: 3, or an active variant or fragment thereof, can further comprise at least one of the following amino acid mutations to generate a catalytically dead enzyme: D413A, E636A, D725A, wherein the indicated mutated amino acid residues correspond to the amino acid residues of the sequence set forth as SEQ ID NO: 3. A Type V RGN comprising the amino acid sequence set forth as SEQ ID NO: 3, or an active variant or fragment thereof, can further comprise two or three of the following amino acid mutations to generate a catalytically dead enzyme: D413A, E636A, D725A, wherein the indicated mutated amino acid residues correspond to the amino acid residues of the sequence set forth as SEQ ID NO: 3.

[0172] A Type V RGN comprising the amino acid sequence set forth as SEQ ID NO: 4, or an active variant or fragment thereof, can further comprise at least one of the following amino acid mutations to generate a catalytically dead enzyme: D396A, E622A, D711A, wherein the indicated mutated amino acid residues correspond to the amino acid residues of the sequence set forth as SEQ ID NO: 4. A Type V RGN comprising the amino acid sequence set forth as SEQ ID NO: 4, or an active variant or fragment thereof, can further comprise two or three of the following amino acid mutations to generate a catalytically dead enzyme: D396A, E622A, D711A, wherein the indicated mutated amino acid residues correspond to the amino acid residues of the sequence set forth as SEQ ID NO: 4. In some embodiments, a catalytically dead RGN polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence set forth as SEQ ID NO: 2687.

[0173] A Type V RGN comprising the amino acid sequence set forth as SEQ ID NO: 4, or an active variant or fragment thereof, can further comprise at least one of the following amino acid mutations to generate a catalytically dead enzyme: D396R, E622R, D711R, wherein the indicated mutated amino acid residues correspond to the amino acid residues of the sequence set forth as SEQ ID NO: 4. In some embodiments, a catalytically dead RGN polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence set forth as any one of SEQ ID NOs: 827, 896, and 979. A Type V RGN comprising the amino acid sequence set forth as SEQ ID NO: 4, or an active variant or fragment thereof, can further comprise two or three of the following amino acid mutations to generate a catalytically dead enzyme: D396R, E622R, D711R, wherein the indicated mutated amino acid residues correspond to the amino acid residues of the sequence set forth as SEQ ID NO: 4.

[0174] III. Fusion Proteins Comprising RNA-guided Nucleases

[0175] The present disclosure provides fusion proteins comprising the presently disclosed RGN polypeptides operably fused to at least one heterologous polypeptide, as well as polynucleotides encoding the fusion proteins. When used to refer to the joining of two protein coding regions (either by fusion or insertion), by “operably linked” or “operably fused” is intended that the coding regions are in the same reading frame, even if one is inserted into another. In some embodiments, polypeptides that are “operably fused” or “operably linked” means that the structure and / or biological activity of each individual peptide is also present in the fusion.

[0176] As used herein, “heterologous”, in reference to a polypeptide that is heterologous to another polypeptide (e.g., an RGN polypeptide), is a polypeptide that is not operably fused to the presently described RGN polypeptides in nature. The heterologous polypeptide can originate from a foreign species or from the same species. The heterologous polypeptide can be in its native form or is substantially modified from its native form in composition and / or genomic locus by deliberate human intervention. The heterologous polypeptide can be any polypeptide, including but not limited to a localization signal, a cell-penetrating domain, a detectable label (e.g., fluorescent protein), a purification tag, a base editing polypeptide (e.g., deaminase), or a polymerase editing polypeptide (e.g., a DNA polymerase, a reverse transcriptase). The heterologous polypeptide can include an effector domain, which can comprise a domain such as a cleavage domain, a deaminase domain, or an expression modulator domain, as further described herein.

[0177] The heterologous polypeptide or effector domain can be located at the N-terminus, the C-terminus, or an internal location of the RGN polypeptide, either directly or indirectly via a peptide linker. Alternatively, the heterologous polypeptide or effector domain can be located in an internal location of the RGN polypeptide (e.g., a base editing polypeptide such as a deaminase or a polymerase editing polypeptide such as a DNA polymerase or a reverse transcriptase), either directly or indirectly via a peptide linker.

[0178] RGNs including those that comprise nuclease activity or that lack nuclease activity can be used to deliver a fused polypeptide, polynucleotide, or small molecule payload to a particular genomic location. In some of these embodiments, the RGN polypeptide or guide RNA can be fused to a detectable label or purification tag to allow for detection of a particular sequence. As a non-limiting example, a nuclease-dead RGN can be fused to a detectable label (e.g. , fluorescent protein) and targeted to a particular sequence associated with a disease to allow for detection of the disease-associated sequence. As another non-limiting example, a nuclease active RGN can be fused to a detectable label (e.g., fluorescent protein) and targeted to a particular sequence associated with a disease to allow for detection of the disease-associated sequence. The detectable label or purification tag can be located at the N-terminus, the C-terminus, or an internal location of the RGN, either directly or indirectly via a linker peptide. In some embodiments, the RGN component of the fusion protein is a nuclease-dead RGN. In some embodiments, the RGN component of the fusion protein is an RGN with nickase activity.

[0179] A detectable label is a molecule that can be visualized or otherwise observed. The detectable label may be fused to the RGN as a fusion protein (e.g., fluorescent protein) or may be a small molecule conjugated to the RGN polypeptide that can be detected visually or by other means. Detectable labels that can be fused to the presently disclosed RGNs as a fusion protein include any detectable protein domain, including but not limited to, a fluorescent protein or a protein domain that can be detected with a specific antibody. Non-limiting examples of fluorescent proteins include green fluorescent proteins (e.g., GFP, EGFP, ZsGreenl) and yellow fluorescent proteins (e g., YFP, EYFP, ZsYellowl). Non-limiting examples of small molecule detectable labels include radioactive labels, such as3H and35S.

[0180] RGN polypeptides of the disclosure can also comprise a purification tag, which is any molecule that can be utilized to isolate a protein or fused protein from a mixture (e.g., biological sample, culture medium). Non-limiting examples of purification tags include biotin, myc, maltose binding protein (MBP), glutathione- S-transferase (GST), and 3X FLAG tag.

[0181] Alternatively, nuclease-dead RGNs can be targeted to particular genomic locations to alter the expression of a desired gene (i.e., target gene). In some embodiments, the binding of a nuclease-dead RGN to a target sequence results in the reduction in expression of the target gene by interfering with the binding of RNA polymerase or transcription factors within the targeted genomic region. In some embodiments, the RGN (e.g., a nuclease-dead RGN) or its complexed guide RNA further comprises an expression modulator that, upon binding to a target sequence within a target gene, serves to either repress or activate the expression of the target gene.

[0182] The effector domain can comprise an expression modulator. In some embodiments, the expression modulator of the fusion protein comprises a transcriptional repressor domain, which interacts with transcriptional control elements and / or transcriptional regulatory proteins, such as RNA polymerases and transcription factors, to reduce or terminate transcription of at least one gene. Transcriptional repressor domains are known in the art and include, but are not limited to, Spl-like repressors, IKB, and Kriippel associated box (KRAB) domains.

[0183] In some embodiments, the expression modulator of the fusion protein comprises a transcriptional activation domain, which interacts with transcriptional control elements and / or transcriptional regulatory proteins, such as RNA polymerases and transcription factors, to increase or activate transcription of at least one gene. Transcriptional activation domains are known in the art and include, but are not limited to, a herpes simplex virus VP 16 activation domain and an NF AT activation domain.

[0184] In some embodiments, the expression modulator of the fusion protein comprises an epigenetic modification domain that modulates the expression of the target sequence or regulated gene through epigenetic mechanisms. In some embodiments, an epigenetic modulator covalently modifies DNA or histone proteins to alter histone structure and / or chromosomal structure without altering the DNA sequence, leading to changes in gene expression (e.g., upregulation or downregulation). Non-limiting examples of epigenetic modifications include acetylation or methylation of lysine residues, arginine methylation, serine and threonine phosphorylation, and lysine ubiquitination and sumoylation of histone proteins, and methylation and hydroxymethylation of cytosine residues in DNA. Non-limiting examples of epigenetic modulators include histone acetyltransferases, histone deacetylases, histone methyltransferases, histone demethylases, DNA methyltransferases, and DNA demethylases.

[0185] The effector domain can comprise a deaminase domain. In some embodiments, the nuclease-dead RGNs or an RGN with nickase activity can be targeted to particular genomic locations to modify the sequence of a target polynucleotide through fusion to a base-editing polypeptide, for example a deaminase polypeptide or active variant or fragment thereof, that directly chemically modifies (e.g., deaminates) a nucleobase, resulting in conversion from one nucleobase to another. A deaminase domain refers to a polypeptide or portion of a polypeptide that can chemically modify (e.g., deaminate) a nucleobase, resulting in conversion from one nucleobase to another. A deaminase or base editing polypeptide can comprise a deaminase domain. In some embodiments, a deaminase and a deaminase domain may be the same polypeptide. The base-editing polypeptide can be fused to the RGN at its amino-terminal (N-terminal) or carboxy-terminal (C-terminal) end. Additionally, the base-editing polypeptide may be fused to the RGN via a peptide linker. “Base editors” are fusion proteins comprising a DNA-targeting polypeptide, such as an RGN, and a base-editing polypeptide, such as a deaminase. Non-limiting examples of deaminases or base editors that are useful for such compositions and methods includes a cytosine deaminase, an adenine deaminase, or a base editor (such as the adenine deaminase or base editor described in Gaudelli et al. (2017) Nature 551:464-471, U.S. Publ. Nos. 2017 / 0121693 and 2018 / 0073012, and International Publ. No. WO 2018 / 027078, or any of the deaminases or base editors disclosed in International Publ. Nos. WO 2020 / 139783, WO 2022 / 056254, WO 2022 / 204093, and WO 2024 / 095245, each of which is herein incorporated by reference in its entirety). In some embodiments, the deaminase that is useful for such presently disclosed compositions and methods is a deaminase disclosed in Table 17 of International Publ. No. WO 2020 / 139783, which is incorporated herein by reference in its entirety. Further, it is known in the art that certain fusion proteins between an RGN and a base-editing enzyme (e.g., cytosine deaminase) may also comprise at least one uracil stabilizing polypeptide that increases the mutation rate of a cytidine, deoxycytidine, or cytosine to a thymidine, deoxythymidine, or thymine in a nucleic acid molecule by a deaminase. Non-limiting examples of uracil stabilizing polypeptides include those disclosed in PCT Publication No. WO 2021 / 217002 and PCT Publication No. WO 2022 / 015969, each of which is herein incorporated by reference in its entirety. The disclosed uracil stabilizing polypeptides include USP2, and a uracil glycosylase inhibitor (UGI) domain, which may increase base editing efficiency. Therefore, a base editor may comprise an RGN described herein or variant thereof, a deaminase, and optionally at least one uracil stabilizing polypeptide, such as UGI or USP2.

[0186] In some embodiments, an RGN may be fused to a polymerase or prime editing polypeptide (e.g., a DNA polymerase or a reverse transcriptase (RT)). Polymerase or prime editing is a versatile and precise genome editing method that directly writes new genetic information into a specified DNA site using a nucleic acid programmable DNA binding protein working in association with a polymerase (described in, e.g., US 11,447,770BI; WO2021072328; WO2021226558; WO2020156575; W02021042047;

[0187] US 11193123; each incorporated by reference in its entirety herein). The polymerase or prime editing system uses an RGN that is a nickase and a DNA polymerase (e.g., RT), and the system is programmed with a polymerase or prime editing (PE) guide RNA (“PEgRNA”). The PEgRNA is a guide RNA that comprises a primer binding site (PBS) and a DNA synthesis template that serves as the template for polymerization of the replacement strand comprising the edit. The PEgRNA specifies the target sequence, and the DNA synthesis template of the PEgRNA is provided by way of an extension engineered onto the guide RNA backbone (e.g., at the 5' or 3' end, or at an internal portion of the guide RNA backbone).

[0188] As used herein, a “polymerase editor” or “PE” refers to a protein or a plurality of proteins comprising a DNA polymerase (e.g., a reverse transcriptase) and a RNA-binding polypeptide (e.g., RGN polypeptide) that, along with a PEgRNA comprising an extension comprising a primer binding site (PBS) and a DNA synthesis template comprising a desired edit, is capable of editing a double -stranded polynucleotide through the replacement of a target sequence using the DNA synthesis template as a template for the DNA polymerase (e.g., a reverse transcriptase). The PE comprising an RGN nickase / DNA polymerase (i.e. a reverse transcriptase) fusion is guided to the target sequence by the PEgRNA and nicks the non-target strand upstream of the sequence to be edited and downstream of the PAM, creating a 3' flap on the non-target strand. The PBS is complementary to the 3' flap of the non-target strand and hybridrization of the PBS and 3' flap of the non-target strand allows for the polymerization of the replacement strand containing the edit using the DNA synthesis template. In some embodiments, a PBS is at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In certain embodiments, the PEgRNA comprises a PBS that is at least 5 (e.g., at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 28, 19, or 20) nucleotides in length. In some embodiments, the PEgRNA may comprise a PBS that is at least 8 nucleotides in length. In some embodiments, the PEgRNA may comprise a PBS that is 9, 11, 12, 13, or 15 nucleotides in length. Hybridrization of the PBS and 3' flap of the non-target strand allows polymerization of the replacement strand containing the edit using the DNA synthesis template in the extension of the PEgRNA. The DNA synthesis template can be at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more nucleotides in length. In certain embodiments, the PEgRNA comprises a DNA synthesis template that is at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, or at least 50 nucleotides in length. In some embodiments, the PEgRNA comprises a DNA synthesis template that is 19, 22, 23, 24, 25, 26, 29, 30, 32, 34, 37, 38, 39, 40, 42, or 46 nucleotides in length. The DNA synthesis template comprises the desired edit, which can be a substitution of one or more nucleotides, a deletion of one or more nucleotides, or an addition of one or more nucleotides.

[0189] The extension of the PEgRNA can be formed from RNA or DNA. In the case of an RNA extension, the polymerase of the polymerase or prime editor can be an RNA-dependent DNA polymerase (such as a reverse transcriptase). In the case of a DNA extension, the polymerase of the polymerase or prime editor may be a DNA-dependent DNA polymerase.

[0190] The replacement strand containing the desired edit (e.g., substitution, deletion, or addition) shares the same sequence as the non-target strand of the target sequence to be edited (with the exception that it includes the desired edit). Through DNA repair and / or replication machinery, the non-target strand of the target sequence is replaced by the newly synthesized replacement strand containing the desired edit. In some cases, polymerase or prime editing may be thought of as a “search-and-replace” genome editing technology since the polymerase or prime editors not only search and locate the desired target sequence to be edited, but at the same time, encode a replacement strand containing a desired edit which is installed in place of the corresponding non-target strand of the target sequence. Thus, in some embodiments, a guide RNA of the disclosure comprises an extension comprising an edit template for polymerase or prime editing. In some embodiments, a polymerase or prime editing polypeptide that can be fused to an RGN includes a DNA polymerase. In certain embodiments, the DNA polymerase is a reverse transcriptase. In certain embodiments, the RGN is a nickase.

[0191] In certain embodiments, an RGN polypeptide and a polymerase editing polypeptide (e.g., RT) of a polymerase editor of the disclosure can be provided in trans instead of as a fusion protein, wherein the RGN polypeptide and the polymerase editing polypeptide (e.g., RT) are separate polypeptides. In some embodiments, the RGN polypeptide and the polymerase editing polypeptide (e.g., RT) are transcribed together and have a sequence encoding a self-cleaving peptide (e.g., 2A peptide such as P2A) in between, such that translation results in two separate polypeptides. Any self-cleaving peptide known in the art can be used in such embodiments, including but not limited to, 2A peptides, which is a class of 18-22 amino acid long peptides that may function through ribosomal skipping during translation. Non-limiting examples of 2A peptides are T2A, P2A, E2A, and F2A.

[0192] In order to reduce the possibility that the edit introduced by a polymerase editor is removed due to mismatch repair of the edited strand, a nicking guide RNA can be used. A “nicking guide RNA” is a guide RNA that targets a sequence within the unedited strand at a site nearby and opposite to the original nick and guides the RGN nickase of the PE system to this unedited strand to introduce a single -stranded nick. The nicking guide RNA can be designed to match the edited sequence introduced by the PEgRNA, but not the original unedited sequence, to ensure that the nicking occurs after the editing event on the non-target strand takes place.

[0193] The effector domain can comprise a cleavage domain. In some embodiments, the RGN fusion protein comprises a cleavage domain, which is any domain that is capable of cleaving a polynucleotide (z.e., RNA, DNA, or RNA / DNA hybrid) and includes, but is not limited to, restriction endonucleases and homing endonucleases, such as Type IIS endonucleases (e.g., FokI) (see, e.g., Belfort et al. (1997) Nucleic Acids Res. 25:3379-3388; Linn et al. (eds.) Nucleases, Cold Spring Harbor Laboratory Press, 1993).

[0194] RGNs that are fused to a heterologous polypeptide or domain can be separated or joined by a linker. The term "linker," as used herein, refers to a chemical group or a molecule linking two molecules or moieties, e.g., a binding domain and a cleavage domain of a nuclease. In some embodiments, a linker joins a gRNA binding domain of an RGN and a base-editing polypeptide, such as a deaminase. In some embodiments, a linker joins a nuclease-dead RGN or RGN nickase and a deaminase. Typically, the linker is positioned between, or flanked by, two groups, molecules, or other moieties and connected to each one via a covalent bond, thus connecting the two. In some embodiments, the linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker is an amino acid or a plurality of amino acids (e.g., a peptide or protein linker). In some embodiments, the linker is 5-100 amino acids in length, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, or 150-200 amino acids in length. Longer or shorter linkers are also contemplated. In some embodiments, a peptide linker comprises at least one NLS. In some embodiments, a peptide linker comprises 2 NLSs. The peptide linker can be operably fused at the N-terminus, the C-terminus, or both the N-terminus and C-terminus of the RGN polypeptide or the heterologous polypeptide (e.g., a base editing polypeptide or a polymerase editing polypeptide). In some embodiments, a peptide linker comprises one or more copies of amino acid sequence GSSG (SEQ ID NO: 1928). In some embodiments, a peptide linker comprises the amino acid sequence set forth as GSSGGSSG (SEQ ID NO: 34). In some embodiments, the peptide linker has an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to SEQ ID NO: 1676 or 1677. In some embodiments, the peptide linker has the amino acid sequence of SEQ ID NO: 1676 or 1677.

[0195] The presently disclosed RGNs can comprise at least one nuclear localization signal (NLS) to enhance transport of the RGN to the nucleus of a cell. Nuclear localization signals are known in the art and generally comprise a stretch of basic amino acids (see, e.g., Lange et al., J. Biol. Chem. (2007) 282:5101- 5105). In some embodiments, the RGN comprises 2, 3, 4, 5, 6 or more nuclear localization signals. The nuclear localization signal(s) can be a heterologous NLS. Non-limiting examples of nuclear localization signals useful for the presently disclosed RGNs are the nuclear localization signals of SV40 Large T-antigen, nucleoplasmin, and c-Myc (see, e.g., Ray et al. (2015) Bioconjug Chem 26(6): 1004-7). In some embodiments, the NLS has an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to SEQ ID NO: 33, 35, 407, or 1927. In some embodiments, the RGN comprises the NLS sequence set forth as SEQ ID NO: 33, 35, 407, or 1927. The RGN can comprise one or more NLS sequences at its N-terminus, C- terminus, or both the N-terminus and C-terminus. Lor example, the RGN can comprise two NLS sequences at the N-terminal region and four NLS sequences at the C- terminal region.

[0196] Other localization signal sequences known in the art that localize polypeptides to particular subcellular location(s) can also be used to target the RGNs, including, but not limited to, plastid localization sequences, mitochondrial localization sequences, and dual-targeting signal sequences that target to both the plastid and mitochondria (see, e.g., Nassoury and Morse (2005) Biochim Biophys Acta 1743:5-19; Kunze and Berger (2015) Front Physiol dx.doi.org / 10.3389 / fphys.2015.00259; Herrmann and Neupert (2003) IUBMB Life 55:219-225; Soil (2002) Curr Opin Plant Biol 5:529-535; Carrie and Small (2013) Biochim Biophys Acta 1833:253-259; Carrie et al. (2009) FEBS J 276: 1187-1195; Silva-Filho (2003) Curr Opin Plant Biol 6:589-595; Peeters and Small (2001) Biochim Biophys Acta 1541:54-63; Murcha et al. (2014) J Exp Bot 65:6301-6335; Mackenzie (2005) Trends Cell Biol 15:548-554; Glaser et al. (1998) Plant Mol Biol 38:311-338).

[0197] The presently disclosed RGNs can comprise at least one cell-penetrating domain that facilitates cellular uptake of the RGN. Cell-penetrating domains are known in the art and generally comprise stretches of positively charged amino acid residues (z.e., polycationic cell-penetrating domains), alternating polar amino acid residues and non-polar amino acid residues (z.e., amphipathic cell -penetrating domains), or hydrophobic amino acid residues (z.e., hydrophobic cell-penetrating domains) (see, e.g., Milletti F. (2012) Drug Discov Today 17:850-860). A non-limiting example of a cell-penetrating domain is the transactivating transcriptional activator (TAT) from the human immunodeficiency virus 1.

[0198] The nuclear localization signal, plastid localization signal, mitochondrial localization signal, dualtargeting localization signal, and / or cell-penetrating domain can be located at the amino-terminus (N- terminus), the carboxyl-terminus (C-terminus), or in an internal location of the RGN.

[0199] IV. Guide RNA

[0200] The present disclosure provides guide RNAs and polynucleotides encoding the same that target an associated RNA-guided nuclease (RGN) to a target sequence. The term “guide RNA” refers to a nucleotide sequence having sufficient complementarity with a target nucleotide sequence to hybridize with the target sequence and direct sequence -specific binding of an associated RGN to the target nucleotide sequence. More specifically, when the target nucleotide sequence is double -stranded as is the case with DNA, the target nucleotide sequence is comprised of a target strand and the non-target strand (which comprises the PAM sequence). In these embodiments, the guide RNA has sufficient complementarity with the target strand of a double-stranded target sequence (e.g., target DNA sequence) such that the guide RNA hybridizes with the target strand and directs sequence-specific binding of an associated RGN to the target sequence (e.g., target DNA sequence). Therefore, in some embodiments, a guide RNA includes a spacerthat is identical to the sequence of the non-target strand except that uracil (U) replaces thymidine (T) in the guide RNA. In embodiments where multiplex gene editing is used and there are multiple guide RNAs, each of the one or more guide RNA has sufficient complementarity with the target strand of a particular target sequence and is capable of hybridizing to the target strand of that target sequence. Thus, “a corresponding target sequence” for a guide RNA refers to the target sequence that the guide RNA has sufficient complementarity with and is capable of hybridizing to.

[0201] An RGN’s respective guide RNA is one or more RNA molecules (generally, one or two), that can bind to the RGN and guide the RGN to bind to a particular target sequence, and in those embodiments wherein the RGN has nickase or nuclease activity, also cleave the target strand and / or the non-target strand. In general, a guide RNA comprises a CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA), although some RGNs do not require a tracrRNA. Specifically, the disclosed RGN and RGN systems include guide RNAs that do not require tracrRNAs. Therefore, in some embodiments, the guide RNA that binds an RGN or that is included in an RGN system of the present disclosure is the crRNA.

[0202] A guide RNA of the disclosure (e.g., a crRNA) can comprise at least one chemical modification. The at least one chemical modification includes: a bridged nucleic acid (BNA) modification; 2'-O-methyl (2'-0-Me) modification; 2'-O-methoxy-ethyl (2'MOE) modification; 2'-fluoro (2'-F) modification; 2'F-4'Ca- OMe modification; 2',4'-di-Ca-OMe modification; 2'-O-methyl 3'phosphorothioate (MS) modification; 2'-O- methyl 3'thiophosphonoacetate (MSP) modification; 2'-O-methyl 3'phosphonoacetate (MP) modification; and phosphorothioate (PS) modification; or a combination thereof. In some embodiments, the BNA comprises a 2', 4' BNA modification. In some embodiments, the 2', 4' BNA modification is selected from the group consisting of: locked nucleic acid (LNA) modification, BNANC[N-Me] modification, 2'-O,4'-C- ethylene bridged nucleic acid (2',4'-ENA) modification, and S-constrained ethyl (cEt) modification. In some embodiments, the 2', 4' BNA is a LNA modification. In some embodiments, the 2', 4' BNA is a cEt modification. In some embodiments, the at least one chemical modification comprises a BNA modification, 2'-0-Me modification, or PS modification. Chemical modifications of spacers, crRNA repeats, crRNAs, tracrRNAs, and guide RNAs are described in WO 2024 / 042489, which is hereby incorporated by reference in its entirety herein.

[0203] The present disclosure provides CRISPR RNAs (crRNAs) or polynucleotides encoding CRISPR RNAs that target an associated RGN to a target sequence. A crRNA comprises a spacer and a CRISPR repeat. The “spacer” has a nucleotide sequence that directly hybridizes with the target strand of a target sequence (e.g., target DNA sequence) of interest. The spacer is engineered to have full or partial complementarity with the target strand of a target sequence of interest. In some embodiments, the spacer can comprise from about 8 nucleotides to about 30 nucleotides, or more. For example, the spacer can be about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, or more nucleotides in length. In some embodiments, the spacer is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more nucleotides in length. In some embodiments, the spacer is about 10 to about 26 nucleotides in length, or about 12 to about 30 nucleotides in length. In some embodiments, the degree of complementarity between a spacer and the target strand of a target sequence (e.g., target DNA sequence), when optimally aligned using a suitable alignment algorithm, is between 50% and 99% or more, including but not limited to about or more than about 50%, about 60%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more. In some embodiments, the degree of complementarity between a spacer and the target strand of a target sequence (e.g., target DNA sequence), when optimally aligned using a suitable alignment algorithm, is 50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more. In some embodiments, the spacer can be identical in sequence to the non-target strand of a target sequence. In some of those embodiments wherein the target sequence is a target DNA sequence, the spacer can be identical in sequence to the non-target strand of the target DNA sequence, with the exception of the thymidines (Ts) in the nontarget strand being replaced by uracils (Us) in the spacer. In particular embodiments, the spacer is free of secondary structure, which can be predicted using any suitable polynucleotide folding algorithm known in the art, including but not limited to mFold (see, e.g., Zuker and Stiegler (1981) Nucleic Acids Res. 9: 133- 148) and RNAfold (see, e.g, Gruber et al. (2008) Cell 106(l):23-24).

[0204] The presently disclosed crRNAs comprise a spacer capable of targeting a bound RGN polypeptide to a target DNA sequence. In some embodiments, a spacer has the nucleotide sequence set forth as any one of SEQ ID NOs: 294-396, 1789-1796, 1798-1880, 1890-1893, and 2082-2125. In some embodiments, the target sequence is in the genome of a eukaryotic cell. The eukaryotic cell can be present ex vivo, in vitro, or in vivo. In some embodiments, the target DNA sequence has the nucleotide sequence set forth as any one of SEQ ID NOs: 183-285, 1684-1691, 1693-1775, 1785-1788, and 2038-2081. In some embodiments, the target sequence is in the genome of a prokaryotic cell.

[0205] Along with a spacer, a crRNA further comprises a CRISPR RNA (crRNA) repeat. Generally, a crRNA repeat comprises a nucleotide sequence that forms a structure, either on its own or in concert with a hybridized tracrRNA, that is recognized by the RGN polypeptide. In the present disclosure, a crRNA repeat can be recognized and bound by an RGN of the disclosure without the crRNA repeat having to hybridize to a tracrRNA. For Type V guide crRNAs, the spacer is 3' of the crRNA repeat. Thus, when considering a Type V crRNA, the crRNA repeat begins at the 5' end of the crRNA, followed by the spacer 3' to the crRNA repeat. The backbone is the part of a guide RNA that does not comprise the spacer. As used herein, the terms ‘crRNA repeat’ and ‘crRNA backbone’ are interchangeable. In various embodiments, the crRNA repeat (i.e. crRNA backbone) can comprise from about 8 nucleotides to about 40 nucleotides, or more. For example, the crRNA repeat (i.e. crRNA backbone) can be about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, or more nucleotides in length. In particular embodiments, the crRNA repeat (i.e. crRNA backbone) is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or more nucleotides in length. In some embodiments, a crRNA repeat (i.e. crRNA backbone) of the disclosure comprises a total length of 25 to 40 nucleotides (nt). In some embodiments, a crRNA repeat (i.e. crRNA backbone) of the disclosure comprises a total length of at most 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, 26 nt, 27 nt, 28 nt, 29 nt, 30 nt, 31 nt, 32 nt, 33 nt, 34 nt, 35 nt, 36 nt, 37 nt, 38 nt, 39 nt, or 40 nt. A crRNA repeat (i.e. crRNA backbone) of the disclosure can include a consensus repeat of the CRISPR array found in the genome of the organism from which the crRNA repeat is obtained.

[0206] In some embodiments, the crRNA repeat (i.e. crRNA backbone) comprises the nucleotide sequence set forth as any one of SEQ ID NOs: 5-8, 1617-1620, 1631, and 1957-1983, or an active variant or fragment thereof, is capable of directing the sequence-specific binding of an associated RGN provided herein to a target sequence of interest. In some embodiments, an active crRNA repeat (i.e. crRNA backbone) variant of a wild-type sequence comprises a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of the nucleotide sequences set forth as SEQ ID NOs: 5-8, 1617-1620, 1631, and 1957-1983. In some embodiments, an active crRNA repeat (i.e. crRNA backbone) fragment of a wild-type sequence comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, or 38 contiguous nucleotides of any one of the nucleotide sequences set forth as SEQ ID NOs: 5-8, 1617-1620, 1631, and 1957-1983.

[0207] In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 5 or differs from SEQ ID NO: 5 by 1 to 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 5 by 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 5 by 4 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 5 by 3 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 5 by 2 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 5 by 1 nucleotide. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 5.

[0208] In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 6 or differs from SEQ ID NO: 6 by 1 to 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 6 by 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 6 by 4 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 6 by 3 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 6 by 2 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 6 by 1 nucleotide. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 6.

[0209] In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 7 or differs from SEQ ID NO: 7 by 1 to 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 7 by 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 7 by 4 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 7 by 3 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 7 by 2 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 7 by 1 nucleotide. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 7.

[0210] In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 8 or differs from SEQ ID NO: 8 by 1 to 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 8 by 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 8 by 4 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 8 by 3 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 8 by 2 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 8 by 1 nucleotide. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 8.

[0211] In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1617 or differs from SEQ ID NO: 1617 by 1 to 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1617 by 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1617 by 4 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1617 by 3 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1617 by 2 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1617 by 1 nucleotide. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1617.

[0212] In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1618 or differs from SEQ ID NO: 1618 by 1 to 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1618 by 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1618 by 4 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1618 by 3 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1618 by 2 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1618 by 1 nucleotide. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1618.

[0213] In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1619 or differs from SEQ ID NO: 1619 by 1 to 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1619 by 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1619 by 4 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1619 by 3 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1619 by 2 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1619 by 1 nucleotide. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1619.

[0214] In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1620 or differs from SEQ ID NO: 1620 by 1 to 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1620 by 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1620 by 4 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1620 by 3 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1620 by 2 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1620 by 1 nucleotide. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1620.

[0215] In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1631 or differs from SEQ ID NO: 1631 by 1 to 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1631 by 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1631 by 4 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1631 by 3 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1631 by 2 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1631 by 1 nucleotide. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1631.

[0216] In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1957 or differs from SEQ ID NO: 1957 by 1 to 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1957 by 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1957 by 4 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1957 by 3 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1957 by 2 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1957 by 1 nucleotide. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1957.

[0217] In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1958 or differs from SEQ ID NO: 1958 by 1 to 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1958 by 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1958 by 4 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1958 by 3 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1958 by 2 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1958 by 1 nucleotide. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1958.

[0218] In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1959 or differs from SEQ ID NO: 1959 by 1 to 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1959 by 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1959 by 4 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1959 by 3 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1959 by 2 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1959 by 1 nucleotide. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1959.

[0219] In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1960 or differs from SEQ ID NO: 1960 by 1 to 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1960 by 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1960 by 4 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1960 by 3 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1960 by 2 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1960 by 1 nucleotide. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1960.

[0220] In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1961 or differs from SEQ ID NO: 1961 by 1 to 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1961 by 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1961 by 4 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1961 by 3 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1961 by 2 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1961 by 1 nucleotide. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1961.

[0221] In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1962 or differs from SEQ ID NO: 1962 by 1 to 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1962 by 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1962 by 4 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1962 by 3 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1962 by 2 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1962 by 1 nucleotide. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1962.

[0222] In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1963 or differs from SEQ ID NO: 1963 by 1 to 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1963 by 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1963 by 4 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1963 by 3 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1963 by 2 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1963 by 1 nucleotide. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1963.

[0223] In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1964 or differs from SEQ ID NO: 1964 by 1 to 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1964 by 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1964 by 4 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1964 by 3 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1964 by 2 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1964 by 1 nucleotide. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1964.

[0224] In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1965 or differs from SEQ ID NO: 1965 by 1 to 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1965 by 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1965 by 4 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1965 by 3 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1965 by 2 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1965 by 1 nucleotide. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1965.

[0225] In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1966 or differs from SEQ ID NO: 1966 by 1 to 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1966 by 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1966 by 4 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1966 by 3 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1966 by 2 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1966 by 1 nucleotide. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1966.

[0226] In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1967 or differs from SEQ ID NO: 1967 by 1 to 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1967 by 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1967 by 4 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1967 by 3 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1967 by 2 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1967 by 1 nucleotide. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1967.

[0227] In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1968 or differs from SEQ ID NO: 1968 by 1 to 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1968 by 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1968 by 4 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1968 by 3 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1968 by 2 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1968 by 1 nucleotide. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1968.

[0228] In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1969 or differs from SEQ ID NO: 1969 by 1 to 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1969 by 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1969 by 4 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1969 by 3 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1969 by 2 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1969 by 1 nucleotide. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1969.

[0229] In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1970 or differs from SEQ ID NO: 1970 by 1 to 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1970 by 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1970 by 4 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1970 by 3 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1970 by 2 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1970 by 1 nucleotide. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1970.

[0230] In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1971 or differs from SEQ ID NO: 1971 by 1 to 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1971 by 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1971 by 4 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1971 by 3 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1971 by 2 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1971 by 1 nucleotide. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1971.

[0231] In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1972 or differs from SEQ ID NO: 1972 by 1 to 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1972 by 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1972 by 4 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1972 by 3 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1972 by 2 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1972 by 1 nucleotide. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1972.

[0232] In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1973 or differs from SEQ ID NO: 1973 by 1 to 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1973 by 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1973 by 4 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1973 by 3 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1973 by 2 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1973 by 1 nucleotide. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1973.

[0233] In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1974 or differs from SEQ ID NO: 1974 by 1 to 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1974 by 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1974 by 4 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1974 by 3 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1974 by 2 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1974 by 1 nucleotide. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1974.

[0234] In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1975 or differs from SEQ ID NO: 1975 by 1 to 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1975 by 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1975 by 4 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1975 by 3 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1975 by 2 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1975 by 1 nucleotide. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1975.

[0235] In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1976 or differs from SEQ ID NO: 1976 by 1 to 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1976 by 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1976 by 4 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1976 by 3 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1976 by 2 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1976 by 1 nucleotide. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1976.

[0236] In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1977 or differs from SEQ ID NO: 1977 by 1 to 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1977 by 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1977 by 4 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1977 by 3 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1977 by 2 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1977 by 1 nucleotide. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1977.

[0237] In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1978 or differs from SEQ ID NO: 1978 by 1 to 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1978 by 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1978 by 4 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1978 by 3 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1978 by 2 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1978 by 1 nucleotide. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1978.

[0238] In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1979 or differs from SEQ ID NO: 1979 by 1 to 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1979 by 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1979 by 4 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1979 by 3 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1979 by 2 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1979 by 1 nucleotide. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1979.

[0239] In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1980 or differs from SEQ ID NO: 1980 by 1 to 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1980 by 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1980 by 4 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1980 by 3 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1980 by 2 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1980 by 1 nucleotide. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1980.

[0240] In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1981 or differs from SEQ ID NO: 1981 by 1 to 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1981 by 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1981 by 4 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1981 by 3 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1981 by 2 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1981 by 1 nucleotide. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1981.

[0241] In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1982 or differs from SEQ ID NO: 1982 by 1 to 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1982 by 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1982 by 4 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1982 by 3 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1982 by 2 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1982 by 1 nucleotide. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1982.

[0242] In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1983 or differs from SEQ ID NO: 1983 by 1 to 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1983 by 5 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1983 by 4 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1983 by 3 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1983 by 2 nucleotides. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has a nucleotide sequence that differs from SEQ ID NO: 1983 by 1 nucleotide. In some embodiments, a crRNA repeat (i.e. crRNA backbone) has the nucleotide sequence set forth as SEQ ID NO: 1983.

[0243] In some embodiments, the crRNA is not naturally-occurring. In some of these embodiments, the specific crRNA repeat (i.e. crRNA backbone) is not linked to the engineered spacer in nature and the crRNA repeat (i.e. crRNA backbone) is considered heterologous to the spacer. In certain embodiments, the spacer is an engineered sequence that is not naturally occurring.

[0244] In some embodiments, because the guide RNA of a Type V-H, V-I, or V-J RGN or RGN system does not require a tracrRNA to function, the crRNA is the guide RNA. A guide RNA (i.e. crRNA) of the disclosure can have the nucleotide sequence set forth as any one of SEQ ID NOs: 39-182, 1405-1412, 1414- 1501, 1556, 1567-1616, 1662-1671, and 2126-2658, or an active variant or fragment thereof. In some embodiments, a guide RNA (i.e. crRNA) of the disclosure can have the nucleotide sequence set forth as any one of SEQ ID NOs: 39-49, 51, 53, 54, 58, 61, 62, 64, 66, 68, 69, 78, 80, 81, 83, 84, 86, 88, 91, 96, 98, 99, 101, 109-128, 130, 132, 133, 135, 137-139, 147-153, 159, 163, 165, 168, 170, 173, 175, 176, 178, 179, and 182, or an active variant or fragment thereof. A guide RNA (i.e. crRNA) of the disclosure can comprise a total length of 35 to 250 nucleotides (nt), or 35 to 220 nt, or 35 to 170 nt, or 35 to 125 nt, or 40 to 100 nt, or 50 nt to 70 nt, or 40 to 70 nt, or 40 to 60 nt. In some embodiments, a guide RNA (i.e. crRNA) of the disclosure can comprise a total length of at most 40 nt, 41 nt, 42 nt, 43 nt, 44 nt, 45 nt, 46 nt, 47 nt, 48 nt, 49 nt, 50 nt. 51 nt, 52 nt, 53 nt, 54 nt, 55 nt, 56 nt, 57 nt, 58 nt, 59 nt, 60 nt, 61 nt, 62 nt, 63 nt, 64 nt, 65 nt, 66 nt, 67 nt, 68 nt, 69 nt, 70 nt, 71 nt, 72 nt, 73 nt, 74 nt, 75 nt, 76 nt, 77 nt, 78 nt, 79 nt, 80 nt, 81 nt, 82 nt, 83 nt, 84 nt, 85 nt, 86 nt, 87 nt, 88 nt, 89 nt, 90 nt, 91 nt, 92 nt, 93 nt, 94 nt, 95 nt, 96 nt, 97 nt, 98 nt, 99 nt, 100 nt, 101 nt, 102 nt, 103 nt, 104 nt, 105 nt, 106 nt, 107 nt, 108 nt, 109 nt, 110 nt, 111 nt, 112 nt, 113 nt, 114 nt,

[0245] 115 nt, 116 nt, 117 nt, 118 nt, 119 nt, 120 nt, 121 nt, 122 nt, 123 nt, 124 nt, 125 nt, 126 nt, 127 nt, 128 nt,

[0246] 129 nt, 130 nt, 131 nt, 132 nt, 133 nt, 134 nt, 135 nt, 136 nt, 137 nt, 138 nt, 139 nt, 140 nt, 141 nt, 142 nt,

[0247] 143 nt, 144 nt, 145 nt, 146 nt, 147 nt, 148 nt, 149 nt, 150 nt, 151 nt, 152 nt, 153 nt, 154 nt, 155 nt, 156 nt,

[0248] 157 nt, 158 nt, 159 nt, 160 nt, 161 nt, 162 nt, 163 nt, 164 nt, 165 nt, 166 nt, 167 nt, 168 nt, 169 nt, 170 nt,

[0249] 180 nt, 190 nt, 200 nt, 205 nt, 210 nt, 215 nt, 220 nt, 225 nt, 230 nt, 235 nt, 240 nt, 245 nt, or 250 nt.

[0250] A guide RNA (i.e. crRNA) of the disclosure can comprise at least one crRNA backbone (bb) and at least one spacer. The at least one crRNA bb can comprise two or more crRNA bb, and the two or more crRNA bb can be different. In some embodiments, the difference comprises a difference in the length of the crRNA bb. In some embodiments, the difference comprises a difference in the sequence of the crRNA bb. In some embodiments, the difference comprises a difference in the length and sequence of the crRNA bb. The at least one crRNA bb can comprise two or more crRNA bb, and the two or more crRNA bb can be the same. In some embodiments, the at least one crRNA bb can comprise two or more crRNA bb, and the two or more crRNA bb can be of the same length. In some embodiments, the at least one crRNA bb can comprise two or more crRNA bb, and the two or more crRNA bb can be of the same sequence. In some embodiments, the at least one crRNA bb can comprise two or more crRNA bb, and the two or more crRNA bb can be of the same length and sequence. The at least one crRNA bb can comprise two or more crRNA bb, and the two or more crRNA bb can include a mix of some crRNA bb that are the same and some crRNA bb that are different. In some embodiments, the at least one crRNA bb can comprise two or more crRNA bb, and the two or more crRNA bb can include a mix of some crRNA bb that share the same lengths and / or sequences and some crRNA bb that are different in length and / or sequence.

[0251] In some embodiments, the guide RNA (i.e. crRNA) is in a format of, from 5’ to 3’, crRNA bb- spacer. In some embodiments, the guide RNA (i.e. crRNA) can comprise two crRNA bb and one spacer in a format of, from 5’ to 3’, crRNA bb-spacer-crRNA bb. In some embodiments, the guide RNA (i.e. crRNA) can comprise three crRNA bb, a first spacer, and a second spacer in a format of, from 5’ to 3’, crRNA bb- first spacer-crRNA bb-second spacer-crRNA bb. One of skill in the art can consider formats with additional crRNA bb and spacers, for example, placing multiple formats of crRNA bb-spacer end to end and ending with a crRNA bb. Some guide formats are shown in FIG. 13A.

[0252] The Type V guide RNAs described herein are self-processing and relatively short in length. These advantages allow the generation of constructs with a tandem arrangement of multiple guide RNAs for editing of multiple targets. Tandem arrays can be created as described in Example 11, where two or more nucleotide sequences, each encoding a crRNA, are placed end to end and operably linked to one promoter. A single polynucleotide encodes a tandem array of crRNAs, which is transcribed as a single transcript and then processed by a type V-H, V-I, or V-J RGN system of the disclosure to multiple single guide RNAs (i.e. crRNAs) which can then target multiple, distinct sequences.

[0253] Two polynucleotide sequences can be considered to be substantially complementary when the two sequences hybridize to each other under stringent conditions. Likewise, an RGN is considered to bind to a particular target sequence within a sequence-specific manner if the guide RNA bound to the RGN binds to a target sequence under stringent conditions. By "stringent conditions" or "stringent hybridization conditions" is intended conditions under which the two polynucleotide sequences will hybridize to each other to a detectably greater degree than to other sequences (e.g. , at least 2-fold over background). Stringent conditions are sequence-dependent and will be different in different circumstances. Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is at least about 30°C for short sequences (e.g., 10 to 50 nucleotides) and at least about 60°C for long sequences (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate) at 37°C, and a wash in IX to 2X SSC (20X SSC = 3.0 M NaCl / 0.3 M trisodium citrate) at 50 to 55°C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37°C, and a wash in 0.5X to IX SSC at 55 to 60°C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37°C, and a wash in 0. IX SSC at 60 to 65°C. Optionally, wash buffers may comprise about 0.1% to about 1% SDS. Duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours. The duration of the wash time will be at least a length of time sufficient to reach equilibrium. The Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched sequence. For DNA-DNA hybrids, the Tm can be approximated from the equation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284: Tm = 81.5°C + 16.6 (log M) + 0.41 (%GC) - 0.61 (% form) - 500 / L; where M is the molarity of monovalent cations, %GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. Generally, stringent conditions are selected to be about 5 °C lower than the thermal melting point (Tm) for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and / or wash at 1, 2, 3, or 4°C lower than the thermal melting point (Tm); moderately stringent conditions can utilize a hybridization and / or wash at 6, 7, 8, 9, or 10°C lower than the thermal melting point (Tm); low stringency conditions can utilize a hybridization and / or wash at 11, 12, 13, 14, 15, or 20°C lower than the thermal melting point (Tm). Using the equation, hybridization and wash compositions, and desired Tm, those of ordinary skill will understand that variations in the stringency of hybridization and / or wash solutions are inherently described. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology — Hybridization with Nucleic Acid Probes, Part I, Chapter 2 (Elsevier, New York); and Ausubel et al., eds. (1995) Current Protocols in Molecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience, New York). See Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York).

[0254] The term “sequence specific” can also refer to the binding of a RGN polypeptide to a target sequence at a greater affinity than binding to a randomized background sequence.

[0255] The guide RNA can be synthesized chemically or via in vitro transcription. Assays for determining sequence -specific binding between an RGN and a guide RNA are known in the art and include, but are not limited to, in vitro binding assays between an expressed RGN and the guide RNA, which can be tagged with a detectable label (e.g., biotin) and used in a pull-down detection assay in which the guide RNA:RGN complex is captured via the detectable label (e.g., with streptavidin beads). A control guide RNA with an unrelated sequence or structure to the guide RNA can be used as a negative control for non-specific binding of the RGN to RNA.

[0256] The guide RNA can be introduced into a target cell, organelle, or embryo as an RNA molecule. In some embodiments, a nucleotide sequence encoding the guide RNA is introduced into a target cell, organelle, or embryo. In some embodiments, the nucleotide sequence encoding a guide RNA is operably linked to a promoter (e.g., an RNA polymerase III promoter). The promoter can be a native promoter or heterologous to the guide RNA-encoding nucleotide sequence. In some embodiments, the guide RNA can be introduced into a target cell, organelle, or embryo as a ribonucleoprotein complex, as described herein, wherein the guide RNA is bound to an RGN polypeptide.

[0257] The guide RNA directs an associated RGN to a particular target nucleotide sequence of interest through hybridization of the guide RNA to the target sequence of interest. The target sequence can be bound (and in some embodiments, cleaved) by an RGN in vitro or in a cell. A target sequence is within a target polynucleotide and can comprise DNA, RNA, or a combination of both and can be single-stranded or double-stranded. A target sequence can be genomic DNA (z.e., chromosomal DNA), plasmid DNA, or an RNA molecule (e.g., messenger RNA, ribosomal RNA, transfer RNA, micro RNA, small interfering RNA). In those embodiments wherein the target sequence is a chromosomal sequence, the chromosomal sequence can be a nuclear, plastid, or mitochondrial chromosomal sequence. In the presently disclosed compositions and methods, the target sequence is within a target nucleic acid molecule that is double-stranded (e.g., a target DNA sequence). In some embodiments, the target sequence is unique in the target genome. In some embodiments, the target sequence comprises a target strand and a non-target strand, and the target sequence (i.e., the sequence on the non-target strand) has the nucleotide sequence set forth as any of SEQ ID NOs: 183-285, 1684-1691, 1693-1775, 1785-1788, and 2038-2081.

[0258] The target sequence is adjacent to a protospacer adjacent motif (PAM) and the non-target strand of the target sequence is the strand that comprises the PAM. The PAM is immediately adjacent to the target sequence and often comprise Ns, which represent any nucleotide. In some embodiments, the PAM comprises about 1 to about 10 Ns, including about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 Ns. In particular embodiments, a PAM comprises 1 to 10 Ns, including 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 Ns. In general, the PAM can be 5' or 3' of the target sequence on its non-target strand. The PAM of the presently disclosed RGNs is immediately 5' of the target sequence on its non-target strand. Generally, the PAM is a consensus sequence of about 3-4 nucleotides, but in particular embodiments it can be 2, 3, 4, 5, 6, 7, 8, 9, or more nucleotides in length.

[0259] In some embodiments, the RGN binds to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) having a nucleotide sequence set forth as any one of SEQ ID NOs: 5-8, 1617-1620, 1631, and 1957-1983, or an active variant or fragment thereof. The RGN systems are described further in Examples 1- 14 and Tables 1-32 of the present specification.

[0260] In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as AYG. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO:

[0261] 1, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as AYG, when bound to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) sequence set forth as SEQ ID NO: 5 or 1631, or an active variant or fragment thereof.

[0262] In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 2, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as ATTN. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO:

[0263] 2, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as ATTN, when bound to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) sequence set forth as SEQ ID NO: 6, or an active variant or fragment thereof. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 3, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as VTTN. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO:

[0264] 3, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as VTTN, when bound to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) sequence set forth as SEQ ID NO: 7, or an active variant or fragment thereof.

[0265] In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 4, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTN. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO:

[0266] 4, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTN, when bound to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) sequence set forth as any one of SEQ ID NOs: 8, and 1617-1620, or an active variant or fragment thereof.

[0267] In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1930, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTN. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1930, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTN, when bound to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) sequence set forth as SEQ ID NO: 1957, or an active variant or fragment thereof.

[0268] In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1931, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as STTN. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1931, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as STTN, when bound to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) sequence set forth as SEQ ID NO: 1958, or an active variant or fragment thereof.

[0269] In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1932, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTN. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1932, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTN, when bound to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) sequence set forth as SEQ ID NO: 1959, or an active variant or fragment thereof.

[0270] In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1933, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as ATTN. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1933, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as ATTN, when bound to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) sequence set forth as SEQ ID NO: 1960, or an active variant or fragment thereof. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1934, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTN. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1934, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTN, when bound to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) sequence set forth as SEQ ID NO: 1961, or an active variant or fragment thereof.

[0271] In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1935, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTH. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1935, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTH, when bound to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) sequence set forth as SEQ ID NO: 1962, or an active variant or fragment thereof.

[0272] In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1936, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTN. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1936, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTN, when bound to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) sequence set forth as SEQ ID NO: 1963, or an active variant or fragment thereof.

[0273] In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1937, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as RTTN. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1937, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as RTTN, when bound to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) sequence set forth as SEQ ID NO: 1964, or an active variant or fragment thereof.

[0274] In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1938, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as VTTN. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1938, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as VTTN, when bound to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) sequence set forth as SEQ ID NO: 1965, or an active variant or fragment thereof.

[0275] In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1939, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTN. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1939, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTN, when bound to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) sequence set forth as SEQ ID NO: 1966, or an active variant or fragment thereof. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1940, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTN. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1940, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTN, when bound to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) sequence set forth as SEQ ID NO: 1967, or an active variant or fragment thereof.

[0276] In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1941, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTN. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1941, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTN, when bound to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) sequence set forth as SEQ ID NO: 1968, or an active variant or fragment thereof.

[0277] In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1942, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTN. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1942, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTN, when bound to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) sequence set forth as SEQ ID NO: 1969, or an active variant or fragment thereof.

[0278] In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1943, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as VTTN. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1943, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as VTTN, when bound to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) sequence set forth as SEQ ID NO: 1970, or an active variant or fragment thereof.

[0279] In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1944, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as ATTN. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1944, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as ATTN, when bound to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) sequence set forth as SEQ ID NO: 1971, or an active variant or fragment thereof.

[0280] In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1945, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as VTTN. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1945, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as VTTN, when bound to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) sequence set forth as SEQ ID NO: 1972, or an active variant or fragment thereof. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1946, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTN. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1946, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTN, when bound to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) sequence set forth as SEQ ID NO: 1973, or an active variant or fragment thereof.

[0281] In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1947, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as VTTN. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1947, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as VTTN, when bound to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) sequence set forth as SEQ ID NO: 1974, or an active variant or fragment thereof.

[0282] In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1948, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTN. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1948, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTN, when bound to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) sequence set forth as SEQ ID NO: 1975, or an active variant or fragment thereof.

[0283] In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1949, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as VTTN. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1949, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as VTTN, when bound to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) sequence set forth as SEQ ID NO: 1976, or an active variant or fragment thereof.

[0284] In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1950, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as RTTN. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1950, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as RTTN, when bound to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) sequence set forth as SEQ ID NO: 1977, or an active variant or fragment thereof.

[0285] In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1951, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as RTYN. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1951, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as RTYN, when bound to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) sequence set forth as SEQ ID NO: 1978, or an active variant or fragment thereof. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1952, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTN. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1952, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTN, when bound to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) sequence set forth as SEQ ID NO: 1979, or an active variant or fragment thereof.

[0286] In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1953, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as ATG. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1953, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as ATG, when bound to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) sequence set forth as SEQ ID NO: 1980, or an active variant or fragment thereof.

[0287] In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1954, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTN. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1954, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTN, when bound to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) sequence set forth as SEQ ID NO: 1981, or an active variant or fragment thereof.

[0288] In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1955, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTN. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1955, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTN, when bound to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) sequence set forth as SEQ ID NO: 1982, or an active variant or fragment thereof.

[0289] In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1956, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTN. In some embodiments, an RGN having the amino acid sequence set forth as SEQ ID NO: 1956, or an active variant or fragment thereof, binds a target nucleotide sequence adjacent to and 3' of a PAM sequence set forth as TTN, when bound to a guide RNA comprising a crRNA repeat (i.e. crRNA backbone) sequence set forth as SEQ ID NO: 1983, or an active variant or fragment thereof.

[0290] It is well-known in the art that PAM sequence specificity for a given nuclease enzyme is affected by enzyme concentration (see, e.g., Karvelis et al. (2015) Genome Biol 16:253), which may be modified by altering the promoter used to express the RGN, or the amount of ribonucleoprotein complex delivered to a target cell, organelle, or embryo.

[0291] Upon recognizing its corresponding PAM sequence, the RGN, if active, may cleave one or both strands of a target sequence at a specific cleavage site. As used herein, a cleavage site comprises particular nucleotides within a target sequence at which the target strand, non-target strand, or both strands of a target sequence are cleaved by an RGN. The cleavage site can comprise the 1stand 2nd, 2ndand 3rd, 3rdand 4th, 4thand 5th, 5thand 6th, 7thand 8th, or 8thand 9thnucleotides from the PAM in either the 5' or 3' direction. The Type V-H, V-I, and V-J RGNs of the present disclosure recognizes a PAM 5' of the cleavage site and thus, the cleavage site can comprise the 1stand 2nd, 2ndand 3rd. 3rdand 4th, 4thand 5th, 5thand 6th, 7thand 8th, or 8thand 9thnucleotides from the PAM in the 3' direction. In some embodiments, the cleavage site may be over 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the PAM in eitherthe 5' or 3' direction. For the Type V-H, V-I, and V-J RGNs and RGN systems of the disclosure, the cleavage site may be over 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides from the PAM in the 3' direction. As RGNs can cleave a target sequence resulting in staggered ends, in some embodiments, the cleavage site is defined based on the distance of the two nucleotides from the PAM on the non-target strand of the target sequence and for the target strand, the distance of the two nucleotides from the complement of the PAM. In some embodiments, a Type V RGN of the disclosure cleaves about 18 nucleotides from the PAM on the target strand. In some embodiments, a Type V RGN of the disclosure cleaves about 23 nucleotides from the complement of the PAM on the target strand.

[0292] Without being bound by any one theory, the distance between the cleavage site for a Type V RGN and the PAM site could potentially allow re-cutting several times after non-homologous end joining (NHEJ) before the PAM / spacer binding is completely destroyed, which may allow more opportunities for homologydependent repair in the case of introducing a donor polynucleotide as part of gene editing.

[0293] V. Polynucleotides encoding RNA-guided nucleases and / or guide RNAs

[0294] The present disclosure provides polynucleotides comprising the presently disclosed guide RNAs (i.e. CRISPR RNAs (crRNAs)) and polynucleotides comprising a nucleotide sequence encoding the presently disclosed RNA-guided nucleases (RGNs) and / or gRNAs (i.e. crRNAs). In various embodiments, the guide RNA (gRNA) that binds an RGN or that is included in an RGN system of the present disclosure is the crRNA, as no tracrRNA molecule is required. Presently disclosed polynucleotides include those comprising or encoding a crRNA comprising a CRISPR RNA (crRNA) repeat (i.e. crRNA backbone) having the nucleotide sequence set forth as any one of SEQ ID NOs: 5-8, 1617-1620, 1631, and 1957-1983, or an active variant or fragment thereof, that when comprised within a guide RNA is capable of directing the sequence -specific binding of an associated RGN to a target sequence of interest. Also provided are polynucleotides that encode an RGN having the amino acid sequence set forth as any one of SEQ ID NOs: 1-4, 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 1087, 1105, 1108, 1116, 1121, 1122, 1123, 1126, 1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173, 1223, 1228, 1304, 1322, 1324, 1328, 1330, 1361, 1400, 1930-1956, 2686, and 2687, and active fragments or variants thereof, that retain the ability to bind to a target sequence in an RNA-guided sequencespecific manner.

[0295] The use of the term "polynucleotide" or “nucleic acid molecule” is not intended to limit the present disclosure to polynucleotides comprising DNA. Those of ordinary skill in the art will recognize that polynucleotides can comprise ribonucleotides (RNA) and combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogues. These include peptide nucleic acids (PNAs), PNA-DNA chimers, locked nucleic acids (LNAs), and phosphothiorate linked sequences. The polynucleotides disclosed herein also encompass all forms of sequences including, but not limited to, single-stranded forms, double -stranded forms, DNA-RNA hybrids, triplex structures, stem-and-loop structures, circular RNA, and the like.

[0296] In some of those embodiments wherein the presently disclosed compositions and methods comprise a polynucleotide encoding an RGN, the polynucleotide is an mRNA (messenger RNA) molecule. An mRNA refers to any polynucleotide which encodes a polypeptide of interest and which is capable of being translated to produce the encoded polypeptide of interest in vitro, in vivo, in situ, or ex vivo. In some embodiments, the basic components of an mRNA molecule include at least a coding region, a 5' untranslated region (UTR), a 3' UTR, a 5' cap and a poly-A tail. A 5' UTR, situated 5' of a coding sequence and transcribed as part of an mRNA, may comprise various regulatory elements, including, e.g., 5' cap structure, G-quadruplex structure (G4), stem-loop structure, and internal ribosome entry sites (IRES), which can control translation initiation of the mRNA. A 3' UTR, situated 3' of a coding sequence and transcribed as part of an mRNA, can be involved in numerous regulatory processes including transcript cleavage, stability and polyadenylation, translation, and mRNA localization. The 3' UTR can serve as a binding site for numerous regulatory proteins and small non-coding RNAs, e.g., microRNAs. A 5' UTR and / or a 3' UTR heterologous to an mRNA originates from an organism or species that is different from that of the mRNA, or if from the same organism or species as the mRNA, is substantially modified from its native form in composition and / or genomic locus by deliberate human intervention.

[0297] In some embodiments, inclusion of a 5' UTR and / or a 3' UTR heterologous to an mRNA encoding an RGN polypeptide of the disclosure improves RGN polypeptide synthesis from the mRNA in a tissue (e.g., liver, or cells in vitro, such as stem cells, hepatocytes or lymphocytes). Heterologous 5' UTRs and / or 3' UTRs may, for example, increase protein synthesis by increasing the time that the mRNA remains in translating polysomes (message stability) and / or the rate at which ribosomes initiate translation on the mRNA (message translation efficiency). Thus, inclusion of a 5' UTR and / or a 3' UTR heterologous to an mRNA encoding an RGN polypeptide of the disclosure can lead to prolonged and / or increased RGN polypeptide synthesis, enabling improved cleavage or modification of a target nucleic acid molecule by the RGN polypeptide. In some embodiments, the enhanced RGN polypeptide synthesis from an mRNA occurs in a tissue-specific manner. Heterologous UTR sequences are described, for example, in US 2023 / 0050143 and US 2017 / 0252461. In some embodiments, an mRNA encoding an RGN useful in the presently disclosed methods and compositions can include one or more structural and / or chemical modifications or alterations which impart useful properties to the polynucleotide. For instance, a useful property of an mRNA includes the lack of a substantial induction of the innate immune response of a cell into which the mRNA is introduced. A “structural” feature or modification is one in which two or more linked nucleotides are inserted, deleted, duplicated, inverted or randomized in an mRNA without significant chemical modification to the nucleotides themselves. Because chemical bonds will necessarily be broken and reformed to effect a structural modification, structural modifications are of a chemical nature and hence are chemical modifications. However, structural modifications will result in a different sequence of nucleotides. Chemical modifications to mRNA can involve inclusion of 5 -methylcytosine, Nl-methyl-pseudouridine, pseudouridine, 2- thiouridine, 4-thiouridine, 5 -methoxyuridine, 2'Fluoroguanosine, 2 'Fluorouridine, 5-bromouridine, 5-(2- carbomethoxyvinyl) uridine, 5-[3(l-E-propenylamino)] uridine, a-thiocytidine, N6-methyladenosine, 5- methylcytidine, N4-acetylcytidine, 5 -formylcytidine, or combinations thereof, in an mRNA.

[0298] The nucleic acid molecules encoding RGNs can be codon optimized for expression in an organism of interest. A "codon-optimized” coding sequence is a polynucleotide coding sequence having its frequency of codon usage designed to mimic the frequency of preferred codon usage or transcription conditions of a particular host cell. Expression in the particular host cell or organism is enhanced as a result of the alteration of one or more codons at the nucleic acid level such that the translated amino acid sequence is not changed. Nucleic acid molecules can be codon optimized, either wholly or in part. Codon tables and other references providing preference information for a wide range of organisms are available in the art (see, e.g., Campbell and Gowri (1990) Plant Physiol. 92 : 1 - 11 for a discussion of plant-preferred codon usage) . Methods are available in the art for synthesizing plant-preferred genes or mammalian (for example human) codon- optimized coding sequences. See, for example, U.S. Patent Nos. 5,380,831, and 5,436,391, and Murray et al. (1989) Nucleic Acids Res. 17:477-498, herein incorporated by reference. Non-limiting examples of codon-optimized coding sequences for the presently disclosed RGNs are set forth as SEQ ID NOs: 29-32, and 2659-2685.

[0299] Polynucleotides encoding the RGNs and / or gRNAs (i.e. crRNAs) provided herein can be provided in expression cassettes for in vitro expression or expression in a cell, organelle, embryo, or organism of interest. The cassette will include 5' and 3' regulatory sequences operably linked to a polynucleotide encoding an RGN and / or gRNA (i.e. crRNA) provided herein that allows for expression of the polynucleotide. The cassette may additionally contain at least one additional gene or genetic element to be cotransformed into the organism. Where additional genes or elements are included, the components are operably linked. The term “operably linked” is intended to mean a functional linkage between two or more elements. For example, an operable linkage between a promoter and a coding region of interest (e.g., region coding for an RGN and / or gRNA (i.e. crRNA)) is a functional link that allows for expression of the coding region of interest. Operably linked elements may be contiguous or non-contiguous. When used to refer to the joining of two protein coding regions, by “operably linked” or “operably fused” is intended that the coding regions are in the same reading frame. In some embodiments, polypeptides that are “operably fused” means that the structure and / or biological activity of each individual peptide is also present in the fusion. Alternatively, the additional gene(s) or element(s) can be provided on multiple expression cassettes. For example, the nucleotide sequence encoding a presently disclosed RGN can be present on one expression cassette, whereas the nucleotide sequence encoding a guide RNA (i.e. crRNA) can be on a separate expression cassette. Such an expression cassette is provided with a plurality of restriction sites and / or recombination sites for insertion of the polynucleotides to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain a selectable marker gene.

[0300] The expression cassette will include in the 5'-3' direction of transcription, a transcriptional (and, in some embodiments, translational) initiation region i.e., a promoter), an RGN- and / or gRNA (i.e. crRNA)- encoding polynucleotide of the invention, and a transcriptional (and in some embodiments, translational) termination region (i. e. , termination region) functional in the cell or organism of interest. The promoters of the disclsoure are capable of directing or driving expression of a coding sequence in a host cell. The regulatory regions (e.g., promoters, transcriptional regulatory regions, and translational termination regions) may be endogenous or heterologous to the host cell or to each other. As used herein, “heterologous” in reference to a sequence is a sequence that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and / or genomic locus by deliberate human intervention. As used herein, a chimeric gene comprises a coding sequence operably linked to a transcription initiation region that is heterologous to the coding sequence.

[0301] Convenient termination regions include ones from simian virus (SV40), human growth hormone (hGH), bovine growth hormone (BGH), and rabbit beta-globin (rbGlob). See also Proudfoot (1991) Cell 64:671-674; Munroe et al. (1990) Gene 91: 151-158; Schek et al. (1992) Molecular and Cellular Biology 12( 12): 5386-5393; Gil and Proudfoot (1987) Cell 49(3):399-406; Goodwin and Rottman (1992) The Journal of Biological Chemistry 267(23): 16330-16334; and Lanoix and Acheson (1988) EMBO J. 7(8): 2515-2522. Additional termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also Guerineau et al. (1991) Mol. Gen. Genet. 262: 141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5: 141-149; Mogen et a / . (1990) Plant Cell 2: 1261-1272; Munroe et al. (1990) Gene 91: 151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; and Joshi et al. (1987) Nucleic Acids Res. 15:9627-9639.

[0302] Additional regulatory signals include, but are not limited to, transcriptional initiation start sites, operators, activators, enhancers, other regulatory elements, ribosomal binding sites, an initiation codon, termination signals, and the like. See, for example, U.S. Pat. Nos. 5,039,523 and 4,853,331; EPO 0480762A2; Sambrook et al. (1992) Molecular Cloning: A Laboratory Manual, ed. Maniatis et al. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), hereinafter "Sambrook 11"; Davis et al., eds. (1980) Advanced Bacterial Genetics (Cold Spring Harbor Laboratory Press), Cold Spring Harbor, N.Y., and the references cited therein. In preparing the expression cassette, the various DNA fragments may be manipulated, so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame. Toward this end, adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions, may be involved.

[0303] A number of promoters can be used in the practice of the disclosure. The promoters can be selected based on the desired outcome. The nucleic acids can be combined with constitutive, inducible, growth stage-specific, cell type-specific, tissue-preferred, tissue-specific, or other promoters for expression in the organism of interest. See, for example, promoters set forth in WO 99 / 43838 and in US Patent Nos: 8,575,425; 7,790,846; 8,147,856; 8,586832; 7,772,369; 7,534,939; 6,072,050; 5,659,026; 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611; herein incorporated by reference.

[0304] Exemplary constitutive promoters for expression in cells of the present disclosure include: an SV40 early promoter; a mouse mammary tumor virus long terminal repeat (LTR) promoter; adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSV) promoter; a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE); a rous sarcoma virus (RSV) promoter; a human ubiquitin C promoter (UBC); a human U6 small nuclear promoter (U6); an enhanced U6 promoter; a human Hl promoter from RNA polymerase III (Hl); a human elongation factor la promoter (EF1A); a human betaactin promoter (ACTB); a human or mouse phosphoglycerate kinase 1 promoter (PGK); a chicken P-Actin promoter coupled with CMV early enhancer (CAGG); a yeast transcription elongation factor promoter (TEF1); and the like. See, for example, Miyagishi et al. (2002) Nature Biotechnology 20:497-500; Xia et al. (2003) Nucleic Acids Res. 31(17):el00-el00; Pasleau et al. ( 1985) Gene 38:227-232; Martin-Gallardo et al. (1988) Gene 70: 51-56; Oellig and Seliger (1990) J Neurosci Res 26: 390-396; Manthorpe et al. (1993) Hum Gene Ther 4: 419-431; Yew et al. (1997) Hum Gene Ther 8: 575-584; Xu et al. (2001) Gene 272: 149-156; Nguyen et al. (2008) J Surg Res 148: 60-66; Costa et al. (2005) Nat Meth. 2:259-260; Lam and Truong (2020) ACS Synth. Biol. 9( 10) :2625-2631.

[0305] For expression in plants, constitutive promoters also include CaMV 35S promoter (Odell et al. (1985) Nature 313:810-812); rice actin (McElroy et al. (1990) Plant Cell 2: 163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12:619-632 and Christensen et a / . (1992) Plant Mol. Biol. 18:675-689); pEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588); and MAS (Velten t a / . (1984) EMBO J. 3:2723-2730).

[0306] Examples of inducible promoters include: stress-regulated promoters such as Adhl promoter, Hsp70 promoter, and Hsp90 promoter (Wurm et al. (1986) roc. Natl. Acad. Sci. USA. 83:5414-5418; Nover L. Heat Shock Response. CRC Press; Boca Raton, FL, USA: 1991); light-inducible promoters such as PPDK promoter and pepcarboxylase promoter; metal-regulated promoters (Mayo et al. (1982) Cell. 29:99-108; Searle et al. (1985) Mol. Cell. Biol. 5: 1480-1489); hormone-responsive promoters including a glucocorticoid-responsive promoter (Hynes et al. (1981) Proc. Natl. Acad. Sci. USA. 78:2038-2042; Klock et al. (1987) Nature. 329:734-736). Chemically regulated promoters include: In2-2 promoter which is safener induced (U.S. Pat. No. 5,364,780); the Axigl promoter which is auxin induced and tapetum specific but also active in callus (PCT US01 / 22169); the steroid-responsive promoters (see, for example, the ERE promoter which is estrogen induced, and the glucocorticoid-inducible promoter in Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88: 10421-10425 and McNellis et al. (1998) Plant J. 14(2):247-257); tetracyclineinducible and tetracycline-repressible promoters (see, for example, Gatz et al. (1991) Mol. Gen. Genet. 227:229-237; Gossen et al. (1993) Trends Biochem Sci. 18:471-475; Gossen and Bujard (1992) Proc. Natl Acad. Sci. USA 89:5547-5551; Zhou et al. (2006) Gene Ther. 13: 1382-1390; and U.S. Pat. Nos. 5,814,618 and 5,789,156); isopropyl-beta-D-thiogalactopyranoside (IPTG)-regulated promoters; and lactose-regulated promoters. Inducible expression can be obtained using operator systems including AlcR / acetaldehyde, ArgR / L-arginine, BirA / biotinyl-AMP, CymR / cumate, EthR / 2-phenylethylbutyrate, HdnoR / 6- hydroxynicotine, HucR / uric acid, MphR(A) / macrolides, PIP / Streptogramins, Rex / NADH, RheA / heat, ScbR / SCBl, TraR / 3-oxo-C8-HSL, and TtgR / phloretin; see, for example, U.S. Patent No. 8,728,759B2; U.S. Patent No. 7,745,592B2; Weber and Fussenegger (2004) Methods Mol. Biol. 267:451-466; Hartenbach et al. (2007) Nucleic Acids Res. 35:el36; Weber et al. (2009) Metab. Eng. 11: 117-124; Weber et al. (2008) Proc. Natl. Acad. Sci. USA. 105:9994-9998; Malphettes et al. (2005) Nucleic Acids Res. 33:el07; Kemmer et al. (20\0) Nat. Biotechnol. 28:355-360; Weber et a / . (2002) Nat. Biotechnol. 20:901-907; Fussenegger et al. (2000) Nat. Biotechnol. 18: 1203-1208; Weber et al. (2006) Metab. Eng. 8:273-280; Weber et a / . (2003) Nucleic Acids Res. 31:e69; Weber et al. (2003) Nucleic Acids Res. 31 :e71 ; Neddermann et al. (2003) EMBO Rep. 4: 159-165; and Gitzinger et a / . (2009) Proc. Natl. Acad. Sci. USA. 106: 10638-10643. Inducible expression can be obtained using protein-protein interaction systems including: rapamycin-induced interaction between FKBP12 (FK506 binding protein 12) and mTOR (Rivera et al. (1996) Nat.

[0307] Med. 2: 1028-1032; Belshaw et al. (1996) Proc. Natl. Acad. Sci. USA. 93:4604-46077); abscisic acid (ABA)-regulated interaction between PYL1 (abscisic acid receptor) and ABI1 (protein phosphatase 2C56) (Liang et al. (2011) Sci. Signal. 4(164):rs2-rs2); and light-induced protein-protein interaction systems (Wang et a / . (2012) Nat. Methods. 9:266-269; Yamada et a / . (2018) Cell. Rep. 25:487-500).

[0308] Tissue-specific or tissue-preferred promoters can be utilized to target expression of an expression construct within a particular tissue. In some embodiments, the tissue-specific or tissue-preferred promoters are active in mammalian tissue. Examples of tissue-specific or tissue-preferred promoters include promoters that initiate transcription preferentially in certain tissues, such as white blood cells (e.g., CD4 T cell), heart, kidney, liver, CNS, eye, pancreas, skeletal muscle, and testis. In some embodiments, the tissue-specific or tissue-preferred promoters are active in plant tissue. Examples of promoters under developmental control in plants include promoters that initiate transcription preferentially in certain tissues, such as leaves, roots, fruit, seeds, or flowers. A "tissue specific" promoter is a promoter that initiates transcription only in certain tissues. Unlike constitutive expression of genes, tissue-specific expression is the result of several interacting levels of gene regulation. As such, promoters from homologous or closely related plant species can be preferable to use to achieve efficient and reliable expression of transgenes in particular tissues. In some embodiments, the expression comprises a tissue-preferred promoter. A "tissue preferred" promoter is a promoter that initiates transcription preferentially, but not necessarily entirely or solely in certain tissues.

[0309] In some embodiments, the nucleic acid molecules encoding an RGN and / or gRNA (i.e. crRNA) comprise a cell type-specific promoter. A "cell type specific" promoter is a promoter that primarily drives expression in certain cell types in one or more organs. Some examples of cells in which cell type specific promoters may be primarily active include, for example, a primary cell, a neuronal cell, a glial cell, an adipocyte, a cardiomyocyte, a smooth muscle cell, a photoreceptor cell, and a retinal ganglia cell. Some examples of plant cells in which cell type specific promoters functional in plants may be primarily active include, for example, BETL cells, vascular cells in roots, leaves, stalk cells, and stem cells. The nucleic acid molecules can also include cell type preferred promoters. A "cell type preferred" promoter is a promoter that primarily drives expression mostly, but not necessarily entirely or solely in certain cell types in one or more organs. Some examples of cells in which cell type preferred promoters may be preferentially active include, for example, a primary cell, a neuron, an adipocyte, a cardiomyocyte, a smooth muscle cell, and a photoreceptor cell. Some examples of plant cells in which cell type preferred promoters functional in plants may be preferentially active include, for example, BETL cells, vascular cells in roots, leaves, stalk cells, and stem cells.

[0310] The nucleic acid sequences encoding the RGNs and / or gRNAs (i.e. crRNAs) can be operably linked to a promoter sequence that is recognized by a phage RNA polymerase for example, for in vitro mRNA synthesis. In such embodiments, the in w / ro-transcribcd RNA can be purified for use in the methods described herein. For example, the promoter sequence can be a T7, T3, or SP6 promoter sequence or a variation of a T7, T3, or SP6 promoter sequence. In such embodiments, the expressed protein and / or RNAs can be purified for use in the methods of genome modification described herein.

[0311] An RNA polymerase II promoter can be operably linked to a polynucleotide encoding an RGN polypeptide of the disclosure for expression of the RGN polypeptide. For example, RNA pol II promoters useful to express an RGN polypeptide of the disclosure include a CMV pol II promoter, a CMV pol II promoter + CMV upstream enhancer, and a CBh pol II promoter. In some embodiments, a Pol II promoter useful for expression of an RGN polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence set forth as SEQ ID NO: 1674 or 1675. In some embodiments, a Pol III promoter useful for expression of one or more gRNA (i.e. crRNA) comprises the amino acid sequence set forth as SEQ ID NO: 1674 or 1675.

[0312] In some embodiments, the polynucleotide encoding the RGN and / or gRNA (i.e. crRNA) can be linked to a polyadenylation signal (e.g, SV40 polyA signal and other signals functional in plants) and / or at least one transcriptional termination sequence. In some embodiments, a polyadenylation signal useful for the disclosure comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence set forth as SEQ ID NO: 1678. In some embodiments, a polyadenylation signal useful for the disclosure comprises the amino acid sequence set forth as SEQ ID NO: 1678. Additionally, the sequence encoding the RGN also can be linked to sequence(s) encoding at least one nuclear localization signal, at least one cell-penetrating domain, and / or at least one signal peptide capable of trafficking proteins to particular subcellular locations, as described elsewhere herein.

[0313] The polynucleotide encoding the RGN and / or gRNA (i.e. crRNA) can be present in a vector or multiple vectors. A “vector” refers to a polynucleotide composition for transferring, delivering, or introducing a nucleic acid into a host cell. Suitable vectors include plasmid vectors, phagemids, cosmids, artificial / mini-chromosomes, transposons, and viral vectors (e.g., lentiviral vectors, adeno-associated viral vectors, baculoviral vector). The vector can comprise additional expression control sequences (e.g., enhancer sequences, Kozak sequences, polyadenylation sequences, transcriptional termination sequences), selectable marker sequences (e.g., antibiotic resistance genes), origins of replication, and the like. Additional information can be found in "Current Protocols in Molecular Biology" Ausubel et al., John Wiley & Sons, New York, 2003 or "Molecular Cloning: A Laboratory Manual" Sambrook & Russell, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 3rd edition, 2001.

[0314] The vector can also comprise a selectable marker gene for the selection of transformed cells. Selectable marker genes are utilized for the selection of transformed cells or tissues. Marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), as well as genes conferring resistance to herbicidal compounds, such as glufosinate ammonium, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D). Marker genes can include genes that allow selection for growth on a particular nutrient or substance, such as dihydrofolate reductase (DHFR; Simonsen and Levinson (1983) Proc. Natl. Acad. Sci. U.S.A. 80:2495-2499), histidinol dehydrogenase (hisD; Hartman and Mulligan (1988) Proc. Natl. Acad. Sci. U.S.A. 85:8047-8051), puromycin-N-acetyl transferase (PAC or puro; de la Luna et al. (1988) Gene 62: 121- 126), thymidine kinase (IK; Littlefield (1964) Science 145:709-710), and xanthine-guanine phosphoribosyltransferase (XGPRT or gpt; Mulligan and Berg (1981) Proc. Natl. Acad. Sci. U.S.A. 78:2072- 2076).

[0315] The expression cassette or vector comprising the polynucleotide encoding an RGN polypeptide can further comprise a polynucleotide encoding a gRNA (i.e. crRNA). The polynucleotide sequence encoding the gRNA (i.e. crRNA) can be operably linked to at least one transcriptional control sequence for expression of the gRNA (i.e. crRNA) in the organism or host cell of interest. Lor example, the polynucleotide encoding the gRNA (i.e. crRNA) can be operably linked to a promoter sequence that is recognized by RNA polymerase III (Pol III). Examples of suitable Pol III promoters include, but are not limited to, mammalian U6, U3, Hl, and 7SK RNA promoters and rice U6 and U3 promoters, such as the RNA Pol III U6 promoter set forth as SEQ ID NO: 38, as well as the promoters disclosed in WO 2022 / 261394, which is herein incorporated by reference in its entirety. In some embodiments, a Pol III promoter useful for expression of one or more gRNA (i.e. crRNA) comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence set forth as SEQ ID NO: 1672 or 1673. In some embodiments, a Pol III promoter useful for expression of one or more gRNA (i.e. crRNA) comprises the amino acid sequence set forth as SEQ ID NO: 1672 or 1673.

[0316] As indicated, expression constructs comprising nucleotide sequences encoding the RGNs and / or gRNAs (i.e. crRNAs) can be used to transform organisms of interest. Methods for transformation involve introducing a nucleotide construct into an organism of interest. By "introducing" is intended to introduce the nucleotide construct to the host cell in such a manner that the construct gains access to the interior of the host cell. The methods of the invention do not require a particular method for introducing a nucleotide construct to a host organism, only that the nucleotide construct gains access to the interior of at least one cell of the host organism. The host cell can be a eukaryotic or prokaryotic cell. In some embodiments, the eukaryotic host cell is a plant cell, a mammalian cell, an avian cell, or an insect cell. In some embodiments, the eukaryotic cell that comprises or expresses a presently disclosed RGN or that has been modified by a presently disclosed RGN is a human cell. In some embodiments, the eukaryotic cell that comprises or expresses a presently disclosed RGN or that has been modified by a presently disclosed RGN is a primary cell. The term "primary cell" refers to a cell isolated directly from a multicellular organism. Primary cells typically have undergone very few population doublings and are therefore more representative of the main functional component of the tissue from which they are derived in comparison to continuous (tumor or artificially immortalized) cell lines. In some cases, primary cells are cells that have been isolated and then used immediately. In other cases, primary cells cannot divide indefinitely and thus cannot be cultured for long periods of time in vitro. In some embodiments, a primary cell is a primary T cell. In some embodiments, the eukaryotic cell that comprises or expresses a presently disclosed RGN or that has been modified by a presently disclosed RGN is a cell of hematopoietic origin, such as an immune cell (i.e., a cell of the innate or adaptive immune system) including but not limited to a B cell, a T cell, a natural killer (NK) cell, a pluripotent stem cell, an induced pluripotent stem cell, a chimeric antigen receptor T (CAR-T) cell, a monocyte, a macrophage, and a dendritic cell. In some embodiments, the eukaryotic cell that comprises or expresses a presently disclosed RGN or that has been modified by a presently disclosed RGN is an ocular cell, muscle cell (e.g., skeletal muscle cell), epithelial cell (e.g., lung epithelial cell), or a diseased cell (e.g., tumor cell).

[0317] Methods for introducing nucleotide constructs into plants and other host cells are known in the art including, but not limited to, stable transformation methods, transient transformation methods, and virus- mediated methods.

[0318] The methods result in a transformed organism, such as a plant, including whole plants, as well as plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, propagules, embryos and progeny of the same. Plant cells can be differentiated or undifferentiated (e.g, callus, suspension culture cells, protoplasts, leaf cells, root cells, phloem cells, pollen). In some embodiments, the presently disclosed methods can result in a transformed organism or cell line derived from these transformed cells.

[0319] "Transgenic organisms" or "transformed organisms" or "stably transformed" organisms or cells or tissues refers to organisms that have incorporated or integrated a polynucleotide encoding an RGN and / or gRNA (i.e. crRNA) of the disclosure. It is recognized that other exogenous or endogenous nucleic acid sequences or DNA fragments may also be incorporated into the host cell. Agrobacterium-and biolistic- mediated transformation remain the two predominantly employed approaches for transformation of plant cells. However, transformation of a host cell may be performed by infection, transfection, microinjection, electroporation, microprojection, biolistics or particle bombardment, electroporation, silica / carbon fibers, ultrasound mediated, PEG mediated, calcium phosphate co-precipitation, polycation DMSO technique, DEAE dextran procedure, and viral mediated, liposome mediated and the like. Viral-mediated introduction of a polynucleotide encoding an RGN and / or gRNA (i.e. crRNA) includes retroviral, lentiviral, adenoviral, and adeno-associated viral mediated introduction and expression, as well as the use of Caulimoviruses (e.g., cauliflower mosaic virus), Geminiviruses (e.g., bean golden yellow mosaic virus or maize streak virus), and RNA plant viruses (e.g., tobacco mosaic virus).

[0320] Transformation protocols as well as protocols for introducing polypeptides or polynucleotide sequences into plants may vary depending on the type of host cell (e.g, monocot or dicot plant cell) targeted for transformation. Methods for transformation are known in the art and include those set forth in US Patent Nos: 8,575,425; 7,692,068; 8,802,934; 7,541,517; each of which is herein incorporated by reference. See, also, Rakoczy-Trojanowska, M. (2002) Cell Mol Biol Lett. 7:849-858; Jones et al. (2005) Plant Methods 1:5; Rivera et al. (2012) Physics of Life Reviews 9:308-345; Bartlett et al. (2008) Plant Methods 4: 1-12; Bates, G.W. (1999) Methods in Molecular Biology 111:359-366; Binns and Thomashow (1988) Annual Reviews in Microbiology 42:575-606; Christou, P. (1992) The Plant Journal 2:275-281; Christou, P. (1995) Euphytica 85: 13-27; Tzfira et al. (2004) TRENDS in Genetics 20:375-383; Yao et al. (2006) Journal of Experimental Botany 5131 131 Zupan and Zambryski (1995) Plant Physiology 107: 1041-1047; Jones et al. (2005) Plant Methods 1:5.

[0321] Transformation may result in stable or transient incorporation of the nucleic acid into the cell. "Stable transformation" is intended to mean that the nucleotide construct introduced into a host cell integrates into the genome of the host cell and is capable of being inherited by the progeny thereof. "Transient transformation" is intended to mean that a polynucleotide is introduced into the host cell and does not integrate into the genome of the host cell.

[0322] Methods for transformation of chloroplasts are known in the art. See, for example, Svab et al. (1990) Proc. Nail. Acad. Sci. USA 87:8526-8530; Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA 90:913-917; Svab and Maliga (1993) EMBO J. 12:601-606. The method relies on particle gun delivery of DNA containing a selectable marker and targeting of the DNA to the plastid genome through homologous recombination. Additionally, plastid transformation can be accomplished by transactivation of a silent plastid-borne transgene by tissue-preferred expression of a nuclear-encoded and plastid-directed RNA polymerase. Such a system has been reported in McBride et al. (1994) Proc. Natl. Acad. Sci. USA 91:7301- 7305.

[0323] The cells that have been transformed may be grown into a transgenic organism, such as a plant, in accordance with conventional ways. See, for example, McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting hybrid having constitutive expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that the polynucleotide encoding an RGN and / or gRNA (i.e. crRNA) is stably maintained and inherited and then seeds harvested to ensure the presence of the polynucleotide encoding an RGN and / or gRNA (i.e. crRNA). In this manner, the present disclosure provides a transformed plant or plant part having a nucleotide construct of the disclosure, for example, an expression cassette of the disclosure, stably incorporated into their genome. Seed having an expression cassette of the disclosure stably incorporated into their genome can be referred to as "transgenic seed" .

[0324] Alternatively, cells that have been transformed may be introduced into an organism. These cells could have originated from the organism, wherein the cells are transformed in an ex vivo approach. These cells can be autologous (originated and returned to the same subject), allogeneic (the donor and recipient subjects are of the same species).

[0325] The sequences provided herein may be used for transformation of any plant species, including, but not limited to, monocots and dicots. Examples of plants of interest include, but are not limited to, com (maize), sorghum, wheat, sunflower, tomato, crucifers, peppers, potato, cotton, rice, soybean, sugarbeet, sugarcane, tobacco, barley, and oilseed rape, Brassica sp., alfalfa, rye, millet, safflower, peanuts, sweet potato, cassava, coffee, coconut, pineapple, citrus trees, cocoa, tea, banana, avocado, fig, guava, mango, olive, papaya, cashew, macadamia, almond, oats, vegetables, ornamentals, and conifers.

[0326] Vegetables include, but are not limited to, tomatoes, lettuce, green beans, lima beans, peas, and members of the genus Curcumis such as cucumber, cantaloupe, and musk melon. Ornamentals include, but are not limited to, azalea, hydrangea, hibiscus, roses, tulips, daffodils, petunias, carnation, poinsettia, and chrysanthemum. In specific embodiments, plants of the present invention are crop plants (for example, maize, sorghum, wheat, sunflower, tomato, crucifers, peppers, potato, cotton, rice, soybean, sugarbeet, sugarcane, tobacco, barley, oilseed rape, etc.).

[0327] As used herein, the term plant includes plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips, anthers, and the like. Grain is intended to mean the mature seed produced by commercial growers for purposes other than growing or reproducing the species. Progeny, variants, and mutants of the regenerated plants are also included within the scope of the invention, provided that these parts comprise the introduced polynucleotides. Further provided is a processed plant product or byproduct that retains the sequences disclosed herein, including for example, soymeal.

[0328] The polynucleotides encoding the RGNs and / or gRNAs (i.e. crRNAs) or comprising the gRNAs (i.e. crRNAs) can be used to transform any eukaryotic species, including but not limited to animals (e.g., mammals, humans, insects, fish, birds, and reptiles), plants, fungi, amoeba, algae, and yeast. In some embodiments, the polynucleotides encoding the RGNs and / or gRNAs (i.e. crRNAs) or comprising the gRNAs (i.e. crRNAs) can be used to transform any prokaryotic species, including but not limited to, archaea and bacteria (e.g., Bacillus sp., Klebsiella sp. Streptomyces sp., Rhizobium sp., Escherichia sp., Pseudomonas sp., Salmonella sp., Shigella sp., Vibrio sp., Yersinia sp., Mycoplasma sp., Agrobacterium, Lactobacillus sp.).

[0329] Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids in mammalian, insect, or avian cells or target tissues. Such methods can be used to administer nucleic acids encoding components of an RGN system to cells in culture, or in a host organism. Non-viral vector delivery systems include DNA plasmids, RNA (e.g. a transcript of a vector described herein), naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as a liposome. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. Non-limiting examples include vectors utilizing Caulimoviruses (e.g., cauliflower mosaic virus), Geminiviruses (e.g., bean golden yellow mosaic virus or maize steak virus), and RNA plant viruses (e.g., tobacco mosaic virus). For a review of gene therapy procedures, see Anderson, Science 256: 808- 813 (1992); Nabel & Feigner, TIBTECH 11:211-217 (1993); Mitani & Caskey, TIBTECH 11: 162-166 (1993); Dillon, TIBTECH 11: 167-175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt, Biotechnology 6(10): 1149-1154 (1988); Vigne, Restorative Neurology and Neuroscience 8:35-36 (1995); Kremer & Perricaudet, British Medical Bulletin 51 ( 1): 31-44 (1995); Haddada et al., in Current Topics in Microbiology and Immunology, Doerfler and Bohm (eds) (1995); and Yu et al., Gene Therapy 1: 13-26 (1994).

[0330] Methods of non-viral delivery of nucleic acids include lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid: nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam ™ and Lipofectin™). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner, WO 91 / 17424; WO 91 / 16024. Delivery can be to cells (e.g. in vitro or ex vivo administration) or target tissues (e.g. in vivo administration). The preparation of lipidmucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to one of skill in the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291- 297 (1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res. 52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

[0331] The use of RNA or DNA viral based systems for the delivery of nucleic acids takes advantage of highly evolved processes for targeting a virus to specific cells in the body and trafficking the viral payload to the nucleus. Viral vectors can be administered directly to patients (in vivo) or they can be used to treat cells in vitro, and the modified cells may optionally be administered to patients (ex vivo). Conventional viral based systems could include retroviral, lentivirus, adenoviral, adeno-associated and herpes simplex virus vectors for gene transfer. Integration in the host genome is possible with the retrovirus, lentivirus, and adeno-associated virus gene transfer methods, often resulting in long term expression of the inserted transgene. Additionally, high transduction efficiencies have been observed in many different cell types and target tissues.

[0332] The tropism of a retrovirus can be altered by incorporating foreign envelope proteins, expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors that are able to transduce or infect non-dividing cells and typically produce high viral titers. Selection of a retroviral gene transfer system would therefore depend on the target tissue. Retroviral vectors are comprised of cis-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum cis-acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression. Widely used retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immuno deficiency virus (SIV), human immuno deficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., J. Viral. 66:2731-2739 (1992); Johann et al., J. Viral. 66: 1635-1640 (1992); Sommnerfelt et al., J. Viral. 176:58-59 (1990); Wilson et al., J. Viral. 63:2374-2378 (1989); Miller et al., J. Viral. 65:2220-2224 (1991); PCT / US94 / 05700).

[0333] In applications where transient expression is preferred, adenoviral based systems may be used. Adenoviral based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. With such vectors, high titer and levels of expression have been obtained. This vector can be produced in large quantities in a relatively simple system. Adeno-associated virus ("AAV") vectors may also be used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368; WO 93 / 24641; Katin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94: 1351 (1994).

[0334] The term "adeno-associated virus" or "AAV" as used herein refers to a member of the class of viruses associated with this name and belonging to the genus dependoparvovirus, family Parvoviridae. Multiple serotypes of this virus are known to be suitable for gene delivery; all known serotypes can infect cells from various tissue types. At least 11, sequentially numbered, have been described. Non-limiting exemplary serotypes useful in the compositions and methods disclosed herein include any of the 11 serotypes (e.g., AAV2, AAV5, AAV6, AAV8), or variant serotypes, e.g., AAV-DJ. AAV is advantageous over other viral vectors for in vivo delivery of genes (e.g., encoding gene editing components) due to their low toxicity and low probability of causing insertional mutagenesis because it typically does not integrate into the host genome. AAV has a packaging limit of about 4.5 to 4.75 Kb.

[0335] Construction of recombinant AAV vectors is described in a number of publications, including U.S. Pat. No. 5, 173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin, et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al., 1. Viral. 63:03822-3828 (1989). Packaging cells are typically used to form virus particles that are capable of infecting a host cell. Such cells include 293 cells, which package adenovirus, and q / J2 cells or PA317 cells, which package retrovirus. A vector of the disclosure can comprise a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 1679-1683. In some embodiments, a vector of the disclosure comprises the nucleotide sequence set forth as any one of SEQ ID NOs: 1679-1683.

[0336] Viral vectors used in gene therapy are usually generated by producing a cell line that packages a nucleic acid vector into a viral particle. The vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host, other viral sequences being replaced by an expression cassette for the polynucleotide(s) to be expressed. The missing viral functions are typically supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically only possess ITR sequences from the AAV genome which are required for packaging and integration into the host genome. Viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences. The cell line may also be infected with adenovirus as a helper. The helper virus promotes replication of the AAV vector and expression of AAV genes from the helper plasmid. The helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV. Additional methods for the delivery of nucleic acids to cells are known to those skilled in the art. See, for example, US20030087817, incorporated herein by reference.

[0337] In some embodiments, a host cell is transiently or non-transiently transfected with one or more vectors described herein. In some embodiments, a cell is transfected as it naturally occurs in a subject. In some embodiments, a cell that is transfected is taken from a subject.

[0338] In some embodiments, a cell that is transfected is a eukaryotic cell. In some embodiments, the eukaryotic cell is an animal cell (e.g., mammals, humans, insects, fish, birds, and reptiles). In some embodiments, a cell that is transfected is a human cell. In some embodiments, a cell that is transfected is a cell of hematopoietic origin, such as an immune cell (i.e., a cell of the innate or adaptive immune system) including but not limited to a B cell, a T cell, a natural killer (NK) cell, a pluripotent stem cell, an induced pluripotent stem cell, a chimeric antigen receptor T (CAR-T) cell, a monocyte, a macrophage, and a dendritic cell.

[0339] In some embodiments, the cell is derived from cells taken from a subject, such as a cell line. In some embodiments, the cell or cell line is prokaryotic. In some embodiments, the cell or cell line is eukaryotic. In further embodiments, the cell or cell line is derived from insect, avian, plant, or fungal species. In some embodiments, the cell or cell line may be mammalian, such as for example human, monkey, mouse, cow, swine, goat, hamster, rat, cat, or dog. A wide variety of cell lines for tissue culture are known in the art. Examples of cell lines include, but are not limited to, C8161, CCRF-CEM, MOLT, mIMCD-3, NHDF, HeLaS3, Huhl, Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panel, PC-3, TF1, CTLL-2, CIR, Rat6, CVI, RPTE, A1O, T24, 182, A375, ARH-77, Calul, SW480, SW620, SKOV3, SK- UT, CaCo2, P388D1, SEM-K2, WEHI- 231, HB56, TIB55, lurkat, 145.01, LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E, MRC5, MEF, Hep G2, HeLa B, HeLa T4. COS, COS-1, COS-6, COS- M6A, BS-C-1 monkey kidney epithelial, BALB / 3T3 mouse embryo fibroblast, 3T3 Swiss, 3T3-L1, 132-d5 human fetal fibroblasts; 10.1 mouse fibroblasts, 293-T, 3T3, 721, 9L, A2780, A2780ADR, A2780cis, A172, A20, A253, A431, A-549, ALC, B16, B35, BCP-I cells, BEAS-2B, bEnd.3, BHK-21, BR 293, BxPC3, C3H-10T1 / 2, C6 / 36, Cal-27, CHO, CHO-7, CHO-IR, CHO-K1, CHO-K2, CHO-T, CHO Dhfr- / -, COR-L23, COR-L23 / CPR, COR-L235010, CORL23 / R23, COS-7, COV-434, CML Tl, CMT, CT26, D17, DH82, DU145, DuCaP, EL4, EM2, EM3, EMT6 / AR1, EMT6 / AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, HEK-293, HeLa, Hepalclc7, HL-60, HMEC, HT-29, lurkat, IY cells, K562 cells, Ku812, KCL22, KG1, KYO1, LNCap, Ma-Mel 1-48, MC-38, MCF-7, MCF-10A, MDA-MB-231, MDA-MB-468, MDA-MB-435, MDCKII, MDCKII, MOR / 0.2R, MONO-MAC 6, MTD-1A, MyEnd, NCI-H69 / CPR, NCI-H69 / LX10, NCI- H69 / LX20, NCI-H69 / LX4, NIH-3T3, NALM-1, NW- 145, OPCN / OPCT cell lines, Peer, PNT-1A / PNT 2, RenCa, RIN-5F, RMA / RMAS, Saos-2 cells, Sf-9, SkBr3, T2, T-47D, T84, THP1 cell line, U373, U87, U937, VCaP, Vero cells, WM39, WT-49, X63, YAC-1, YAR, and transgenic varieties thereof. Cell lines are available from a variety of sources known to those with skill in the art (see, e.g., the American Type Culture Collection (ATCC) (Manassas, Va.)).

[0340] In some embodiments, a cell transfected with one or more nucleic acid molecules or vectors described herein is used to establish a new cell line comprising one or more vector-derived sequences. In some embodiments, a cell transiently transfected with the components of an RGN system as described herein (such as by transient transfection of one or more vectors, or transfection with RNA), and modified through the activity of an RGN system, is used to establish a new cell line comprising cells containing the modification but lacking any other exogenous sequence. In some embodiments, cells transiently or non- transiently transfected with one or more vectors described herein, or cell lines derived from such cells are used in assessing one or more test compounds.

[0341] In some embodiments, one or more vectors described herein are used to produce a non-human transgenic animal or transgenic plant. In some embodiments, the transgenic animal is an insect. In further embodiments, the insect is an insect pest, such as a mosquito or tick. In some embodiments, the insect is a plant pest, such as a com rootworm or a fall armyworm. In some embodiments, the transgenic animal is a bird, such as a chicken, turkey, goose, or duck. In some embodiments, the transgenic animal is a mammal, such as a human, mouse, rat, hamster, monkey, ape, rabbit, swine, cow, horse, goat, sheep, cat, or dog.

[0342] VI. Variants and Fragments of Polypeptides and Polynucleotides

[0343] The present disclosure provides active variants and fragments of an RNA-guided nuclease (RGN) having the amino acid sequence set forth as SEQ ID NOs: 1-4, 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 1087, 1105, 1108, 1116, 1121, 1122, 1123, 1126, 1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173, 1223, 1228, 1304, 1322, 1324,

[0344] 1328, 1330, 1361, 1400, 1930-1956, 2686, and 2687, and the corresponding CRISPR repeats and gRNAs. In some embodiments, an active variant or fragment of an RGN disclosed herein includes a Type V-H, a Type V-I, or a Type V-J RGN. In some embodiments, the disclosure provides: active variants and fragments of an RGN having the amino acid sequence set forth as any one of SEQ ID NOs: 1-4, 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 1087, 1105, 1108, 1116, 1121, 1122, 1123, 1126, 1132,

[0345] 11...

Claims

THAT WHICH IS CLAIMED:

1. A nucleic acid molecule comprising a polynucleotide encoding an RNA-guided nuclease (RGN) polypeptide, wherein said polynucleotide comprises a nucleotide sequence encoding an RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as any one of SEQ ID NOs: 1-4, 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 1087, 1105, 1108, 1116, 1121, 1122, 1123, 1126,1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173,1223, 1228, 1304, 1322, 1324, 1328, 1330, 1361, 1400, 1930-1956, 2686, and 2687.

2. The nucleic acid molecule of claim 1, wherein said RGN polypeptide comprises the amino acid sequence set forth as any one of SEQ ID NOs: 1-4, 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 1087, 1105, 1108, 1116, 1121, 1122, 1123, 1126,1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173,1223, 1228, 1304, 1322, 1324, 1328, 1330, 1361, 1400, 1930-1956, 2686, and 2687.

3. The nucleic acid molecule of claim 1, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1, and wherein the amino acid residue at one or more of amino acid positions 517, 530, 538, 548, 551, 552, 553, 568, 570, 574, 577, 611, 677, 789, 790, and 801 of said RGN polypeptide differs from the corresponding amino acid residue of SEQ ID NO: 1.

4. The nucleic acid molecule of claim 3, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1, and wherein the amino acid residue(s) at one or more of amino acid positions 517, 530, 538, 548, 551, 552, 553, 568, 570, 574, 577, 611, 677, 789, 790, and 801 of said RGN polypeptide is a positively charged amino acid residue.

5. The nucleic acid molecule of claim 3 or 4, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1, and wherein the amino acid residue(s) at one or more of amino acid positions 517, 530, 538, 548, 551, 552, 553, 568, 570, 574, 577, 611, 677, 789, 790, and 801 of said RGN polypeptide is an arginine (R).

6. The nucleic acid molecule of claim 5, wherein said RGN polypeptide comprises an amino acid sequence having 100% sequence identity to the amino acid sequence selected from:(a) the amino acid sequence set forth as SEQ ID NO: 450;(b) the amino acid sequence set forth as SEQ ID NO: 463;(c) the amino acid sequence set forth as SEQ ID NO: 471;(d) the amino acid sequence set forth as SEQ ID NO: 481;(e) the amino acid sequence set forth as SEQ ID NO: 484;(f) the amino acid sequence set forth as SEQ ID NO: 485;(g) the amino acid sequence set forth as SEQ ID NO: 486;(h) the amino acid sequence set forth as SEQ ID NO: 500;(i) the amino acid sequence set forth as SEQ ID NO: 502;(j) the amino acid sequence set forth as SEQ ID NO: 505;(k) the amino acid sequence set forth as SEQ ID NO: 508;(l) the amino acid sequence set forth as SEQ ID NO: 539;(m) the amino acid sequence set forth as SEQ ID NO: 602;(n) the amino acid sequence set forth as SEQ ID NO: 707;(o) the amino acid sequence set forth as SEQ ID NO: 708; and(p) the amino acid sequence set forth as SEQ ID NO: 717.

7. The nucleic acid molecule of claim 1, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1, and wherein the amino acid residues are arginines (Rs) at a combination of amino acid positions selected from the following combinations of amino acid positions of said RGN polypeptide:(a) amino acid positions 517, 530, 552, and 677;(b) amino acid positions 530, 538, 552, and 801;(c) amino acid positions 530, 552, 677, and 801;(d) amino acid positions 530, 577, and 790;(e) amino acid positions 517, 530, 538, 552, and 677;(f) amino acid positions 530, 538, 677, and 801;(g) amino acid positions 538, 552, 677, and 801;(h) amino acid positions 517, 530, 538, and 552;(i) amino acid positions 517 and 530;(j) amino acid positions 530, 538, and 552;(k) amino acid positions 517, 530, 538, and 677;(l) amino acid positions 517, 552, 677, and 801;(m) amino acid positions 530, 677, and 801;(n) amino acid positions 517, 530, 677, and 801;(o) amino acid positions 517, 530, and 677;(p) amino acid positions 530, 538, 552, and 677;(q) amino acid positions 517, 552, and 677; and(r) amino acid positions 517, 530, 552, 677, and 801.

8. The nucleic acid molecule of claim 7, wherein said RGN polypeptide comprises an amino acid sequence having 100% sequence identity to the amino acid sequence selected from:(a) the amino acid sequence set forth as SEQ ID NO: 1072;(b) the amino acid sequence set forth as SEQ ID NO: 1080;(c) the amino acid sequence set forth as SEQ ID NO: 1082;(d) the amino acid sequence set forth as SEQ ID NO: 1105;(e) the amino acid sequence set forth as SEQ ID NO: 1084;(f) the amino acid sequence set forth as SEQ ID NO: 1081;(g) the amino acid sequence set forth as SEQ ID NO: 1083;(h) the amino acid sequence set forth as SEQ ID NO: 1069;(i) the amino acid sequence set forth as SEQ ID NO: 1034;(j) the amino acid sequence set forth as SEQ ID NO: 1059;(k) the amino acid sequence set forth as SEQ ID NO: 1070;(l) the amino acid sequence set forth as SEQ ID NO: 1078;(m) the amino acid sequence set forth as SEQ ID NO: 1064;(n) the amino acid sequence set forth as SEQ ID NO: 1074;(o) the amino acid sequence set forth as SEQ ID NO: 1051;(p) the amino acid sequence set forth as SEQ ID NO: 1079;(q) the amino acid sequence set forth as SEQ ID NO: 1056; and(r) the amino acid sequence set forth as SEQ ID NO: 1087.

9. The nucleic acid molecule of claim 1, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1, and wherein the amino acid residues are arginines (Rs) at a combination of amino acid positions selected from the following combinations of amino acid positions of said RGN polypeptide :(a) amino acid positions 517, 530, 552, and 677;(b) amino acid positions 530, 538, 552, and 801; and(c) amino acid positions 530, 552, 677, and 801.

10. The nucleic acid molecule of claim 9, wherein said RGN polypeptide comprises an amino acid sequence having 100% sequence identity to the amino acid sequence selected from:(a) the amino acid sequence set forth as SEQ ID NO: 1072;(b) the amino acid sequence set forth as SEQ ID NO: 1080; and(c) the amino acid sequence set forth as SEQ ID NO: 1082.

11. The nucleic acid molecule of claim 1, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4, and wherein the amino acid residue(s) at one or more of amino acid positions 377,378, 438, 625, 638, 641, 645, 691, 692, 695, 698, 702, 707, 747, and 755 of said RGN polypeptide differs from the corresponding amino acid residue of SEQ ID NO: 4.

12. The nucleic acid molecule of claim 11, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4, and wherein the amino acid residue(s) at one or more of amino acid positions 377, 378, 438, 625, 638, 641, 645, 691, 692, 695, 698, 702, 707, 747, and 755 of said RGN polypeptide is a positively charged amino acid residue.

13. The nucleic acid molecule of claim 11 or 12, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4, and wherein the amino acid residue(s) at one or more of amino acid positions 377, 378, 438, 625, 638, 641, 645, 691, 692, 695, 698, 702, 707, 747, and 755 of said RGN polypeptide is an R.

14. The nucleic acid molecule of claim 13, wherein said RGN polypeptide comprises an amino acid sequence having 100% sequence identity to the amino acid sequence selected from:(a) the amino acid sequence set forth as SEQ ID NO: 809;(b) the amino acid sequence set forth as SEQ ID NO: 810;(c) the amino acid sequence set forth as SEQ ID NO: 866;(d) the amino acid sequence set forth as SEQ ID NO: 898;(e) the amino acid sequence set forth as SEQ ID NO: 909;(f) the amino acid sequence set forth as SEQ ID NO: 912;(g) the amino acid sequence set forth as SEQ ID NO: 916;(h) the amino acid sequence set forth as SEQ ID NO: 960;(i) the amino acid sequence set forth as SEQ ID NO: 961;(j) the amino acid sequence set forth as SEQ ID NO: 963;(k) the amino acid sequence set forth as SEQ ID NO: 966;(l) the amino acid sequence set forth as SEQ ID NO: 970;(m) the amino acid sequence set forth as SEQ ID NO: 975;(n) the amino acid sequence set forth as SEQ ID NO: 1012; and(o) the amino acid sequence set forth as SEQ ID NO: 1019.

15. The nucleic acid molecule of claim 1, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4, and wherein the amino acid residues are arginines (Rs) at a combination of amino acid positions selected from the following combinations of amino acid positions of said RGN polypeptide:(a) amino acid positions 377, 625, and 698;(b) amino acid positions 377, 625, 638, 698, and 747;(c) amino acid positions 377, 625, 691, 698, and 747;(d) amino acid positions 377, 638, 691, and 747;(e) amino acid positions 377, 638, 691, 698, and 747;(f) amino acid positions 377, 625, and 691;(g) amino acid positions 377, 625, 698, and 747;(h) amino acid positions 377, 638, 691, and 698;(i) amino acid positions 377, 625, and 638;(j) amino acid positions 377, 638, 698, and 747;(k) amino acid positions 377, 625, 638, 691, and 698;(l) amino acid positions 625, 638, and 698;(m) amino acid positions 377, 625, 691, and 698;(n) amino acid positions 377, 638, and 698;(o) amino acid positions 625, 691, 698, and 747;(p) amino acid positions 638 and 698; and(q) amino acid positions 377 and 691.

16. The nucleic acid molecule of claim 15, wherein said RGN polypeptide comprises an amino acid sequence having 100% sequence identity to the amino acid sequence selected from:(a) the amino acid sequence set forth as SEQ ID NO: 1123;(b) the amino acid sequence set forth as SEQ ID NO: 1158;(c) the amino acid sequence set forth as SEQ ID NO: 1159;(d) the amino acid sequence set forth as SEQ ID NO: 1148;(e) the amino acid sequence set forth as SEQ ID NO: 1160;(f) the amino acid sequence set forth as SEQ ID NO: 1122;(g) the amino acid sequence set forth as SEQ ID NO: 1146;(h) the amino acid sequence set forth as SEQ ID NO: 1147;(i) the amino acid sequence set forth as SEQ ID NO: 1121;(j) the amino acid sequence set forth as SEQ ID NO: 1149;(k) the amino acid sequence set forth as SEQ ID NO: 1156;(l) the amino acid sequence set forth as SEQ ID NO: 1132;(m) the amino acid sequence set forth as SEQ ID NO: 1144;(n) the amino acid sequence set forth as SEQ ID NO: 1126;(o) the amino acid sequence set forth as SEQ ID NO: 1154;(p) the amino acid sequence set forth as SEQ ID NO: 1116; and(q) the amino acid sequence set forth as SEQ ID NO: 1108.

17. The nucleic acid molecule of claim 1, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4, and wherein the amino acid residue(s) at one or more of amino acid positions 685,691, 692, 695, 698, 702, 707, 736, 740, 743, 747, 751, 752, 753, 755, 757, 758, 760, 764, and 766 of said RGN polypeptide differs from the corresponding amino acid residue of SEQ ID NO: 4.

18. The nucleic acid molecule of claim 17, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4, and wherein the amino acid residue(s) at one or more of amino acid positions 685, 691, 692, 695, 698, 702, 707, 736, 740, 743, 747, 751, 752, 753, 755, 757, 758, 760, 764, and 766 of said RGN polypeptide is a positively charged amino acid residue.

19. The nucleic acid molecule of claim 17 or 18, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4, and wherein the amino acid residue(s) at one or more of amino acid positions 685, 691, 692, 695, 698, 702, 707, 736, 740, 743, 747, 751, 752, 753, 755, 757, 758, 760, 764, and 766 of said RGN polypeptide is an R or a lysine (K).

20. The nucleic acid molecule of claim 1, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4, and wherein the amino acid residues are arginines (Rs) and / or lysines (Ks) at a combination of amino acid positions selected from the following combinations of amino acid positions of said RGN polypeptide:(a) amino acid positions 736, 743, 752, 753, 755, 757, 758, and 760;(b) amino acid positions 740, 747, 751, 753, 755, 760, 764, and 766;(c) amino acid positions 685, 691, and 698; and(d) amino acid positions 685, 692, 695, 702, and 707.

21. The nucleic acid molecule of claim 20, wherein said RGN polypeptide comprises an amino acid sequence having 100% sequence identity to the amino acid sequence selected from:(a) the amino acid sequence set forth as SEQ ID NO: 1168;(b) the amino acid sequence set forth as SEQ ID NO: 1171;(c) the amino acid sequence set forth as SEQ ID NO: 1169; and(d) the amino acid sequence set forth as SEQ ID NO: 1170.

22. The nucleic acid molecule of claim 1, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4, and wherein said RGN polypeptide comprises a deletion of amino acid residues corresponding to amino acid residues 751-769 of SEQ ID NO: 4.

23. The nucleic acid molecule of claim 22, wherein said RGN polypeptide comprises the amino acid sequence set forth as SEQ ID NO: 1173.

24. The nucleic acid molecule of claim 1, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1, and wherein one or more of amino acid positions 293, 294, 359, 360, 361, 401, 402,403, 596, and 862 of said RGN polypeptide comprises substitution or insertion of one or more glycines (G) immediately N-terminal to, at, or immediately C-terminal to said amino acid position.

25. The nucleic acid molecule of claim 24, wherein said substitution or insertion of one or more G comprises substitution or insertion of one, two, or three G.

26. The nucleic acid molecule of claim 24 or 25, wherein said substitution or insertion of one or more G is selected from:(a) an insertion of three G between amino acid positions 293 and 294;(b) an insertion of two G between amino acid positions 359 and 360;(c) a substitution of G at amino acid position 361;(d) an insertion of one G between amino acid positions 402 and 403;(e) a substitution of G at amino acid position 401 ;(f) a substitution of G at amino acid position 596; and(g) a substitution of G at amino acid position 862.

27. The nucleic acid molecule of claim 26, wherein said RGN polypeptide comprises an amino acid sequence having 100% sequence identity to the amino acid sequence selected from:(a) the amino acid sequence set forth as SEQ ID NO: 1304;(b) the amino acid sequence set forth as SEQ ID NO: 1322;(c) the amino acid sequence set forth as SEQ ID NO: 1324;(d) the amino acid sequence set forth as SEQ ID NO: 1328;(e) the amino acid sequence set forth as SEQ ID NO: 1330;(f) the amino acid sequence set forth as SEQ ID NO: 1361; and(g) the amino acid sequence set forth as SEQ ID NO: 1400.

28. The nucleic acid molecule of claim 1, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4, and wherein one or more of amino acid positions 566, 567, and 584 of said RGN polypeptide comprises substitution or insertion of one or more G immediately N-terminal to, at, or immediately C-terminal to said amino acid position.

29. The nucleic acid molecule of claim 28, wherein said substitution or insertion of one or more G comprises substitution or insertion of one, two, or three G.

30. The nucleic acid molecule of claim 28 or 29, wherein said substitution or insertion of one or more G is selected from: (a) a substitution of G at amino acid position 566 and a substitution of G at amino acid position 567; and (b) a substitution of G at amino acid position 584.

31. The nucleic acid molecule of claim 30, wherein said RGN polypeptide comprises the amino acid sequence selected from: (a) the amino acid sequence set forth as SEQ ID NO: 1223; and (b) the amino acid sequence set forth as SEQ ID NO: 1228.

32. The nucleic acid molecule of any one of claims 1-31, wherein said polynucleotide encoding an RGN polypeptide is operably linked to a promoter heterologous to said polynucleotide.

33. The nucleic acid molecule of any one of claims 1-31, wherein said polynucleotide encoding an RGN polypeptide is an mRNA comprising a heterologous 5 ’ untranslated region (UTR) and / or a heterologous 3 ’ UTR.

34. The nucleic acid molecule of any one of claims 1-33, wherein said RGN polypeptide is capable of binding a target sequence in a target nucleic acid molecule in an RNA-guided sequence specific manner when bound to a guide RNA (gRNA), wherein said target sequence comprises a target strand and a non-target strand, and wherein said gRNA is capable of hybridizing to the target strand of the target sequence.

35. The nucleic acid molecule of claim 34, wherein said target sequence is located adjacent to and 3' of a protospacer adjacent motif (PAM).

36. The nucleic acid molecule of claim 35, wherein a) the RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO: 1 recognizes the PAM having a consensus nucleotide sequence set forth as AYG, wherein the PAM is adjacent to and 5' of the target sequence; b) the RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO: 2, 1933, or 1944, recognizes the PAM having a consensus nucleotide sequence set forth as ATTN, wherein the PAM is adjacent to and 5' of the target sequence; c) the RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO: 3, 1938, 1943, 1945, 1947, or 1949, recognizes the PAM having a consensus nucleotide sequence set forth as VTTN, wherein the PAM is adjacent to and 5' of the target sequence; d) the RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO: 4, 1930, 1932, 1934, 1936, 1939, 1940, 1941, 1942, 1946, 1948, 1952, 1954, 1955, or 1956, recognizes the PAM having a consensus nucleotide sequence set forth as TTN, wherein the PAM is adjacent to and 5' of the target sequence: e) the RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO: 1931 recognizes the PAM having a consensus nucleotide sequence set forth as STTN, wherein the PAM is adjacent to and 5' of the target sequence; f) the RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO: 1935 recognizes the PAM having a consensus nucleotide sequence set forth as TTH, wherein the PAM is adjacent to and 5' of the target sequence; g) the RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO: 1937 or 1950 recognizes the PAM having a consensus nucleotide sequence set forth as RTTN, wherein the PAM is adjacent to and 5' of the target sequence;h) the RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO: 1951 recognizes the PAM having a consensus nucleotide sequence set forth as RTYN, wherein the PAM is adjacent to and 5' of the target sequence; and i) the RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO: 1953 recognizes the PAM having a consensus nucleotide sequence set forth as ATG, wherein the PAM is adjacent to and 5' of the target sequence.

37. The nucleic acid molecule of any one of claims 34-36, wherein the gRNA does not comprise a trans-activating CRISPR RNA (tracrRNA).

38. The nucleic acid molecule of any one of claims 34-37, wherein the gRNA comprises a total length of 35-250 nucleotides (nt), or 35-170 nt, or 35-125 nt, or 40-100 nt, or 40-70 nt, or 40- 60 nt.

39. The nucleic acid molecule of any one of claims 34-38, wherein the gRNA comprises a crRNA backbone comprising a total length of at most 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, 26 nt, 27 nt, 28 nt, 29 nt, 30 nt, 31 nt, 32 nt, 33 nt, 34 nt, 35 nt, 36 nt, 37 nt, 38 nt, 39 nt, or 40 nt.

40. The nucleic acid molecule of any one of claims 1-39, wherein the nucleotide sequence encoding the RGN polypeptide comprises any one of SEQ ID NOs: 29-32, and 2659-2685.

41. The nucleic acid molecule of any one of claims 34-40, wherein said RGN polypeptide is capable of cleaving said target nucleic acid molecule upon binding.

42. The nucleic acid molecule of claim 41, wherein said RGN polypeptide is capable of generating a double-stranded break.

43. The nucleic acid molecule of claim 1, wherein said RGN polypeptide is nuclease inactive.

44. The nucleic acid molecule of claim 43, wherein said RGN polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to any one of SEQ ID NOs: 408, 597, 678, 827, 896, 979, 2686, and 2687.

45. The nucleic acid molecule of any one of claims 1-44, wherein the RGN polypeptide is operably fused to a base -editing polypeptide.

46. The nucleic acid molecule of claim 45, wherein the base-editing polypeptide comprises a deaminase.

47. The nucleic acid molecule of any one of claims 1-44, wherein the RGN polypeptide is operably fused to a polymerase editing polypeptide.

48. The nucleic acid molecule of claim 47, wherein said polymerase editing polypeptide comprises a DNA polymerase.

49. The nucleic acid molecule of claim 47, wherein said polymerase editing polypeptide comprises a reverse transcriptase.

50. The nucleic acid molecule of any one of claims 1-44, wherein the RGN polypeptide is operably fused to an effector domain.

51. The nucleic acid molecule of claim 50, wherein the effector domain is operably fused at the N-terminus or the C-terminus of the RGN polypeptide.

52. The nucleic acid molecule of claim 50, wherein the effector domain is operably fused at an internal location of the RGN polypeptide.

53. The nucleic acid molecule of any one of claims 50-52, wherein the effector domain comprises a cleavage domain, a deaminase domain, or an expression modulator domain.

54. The nucleic acid molecule of claim 53, wherein the expression modulator domain comprises a trancriptional activation domain, a transcriptional repressor domain, or an epigenetic modification domain.

55. The nucleic acid molecule of any one of claims 1-54, wherein the RGN polypeptide comprises one or more nuclear localization signals (NLS).

56. The nucleic acid molecule of claim 55, wherein the one or more NLS comprises an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to any one of SEQ ID NOs: SEQ ID NOs: 33, 35, 407, and 1927.

57. The nucleic acid molecule of any one of claims 1-56, wherein the nucleotide sequence encoding the RGN polypeptide is codon optimized for expression in a eukaryotic cell.

58. A vector comprising the nucleic acid molecule of any one of claims 1-57.

59. The vector of claim 58, wherein the vector further comprises at least one nucleotide sequence encoding a guide RNA (gRNA).

60. The vector of claim 59, wherein said at least one nucleotide sequence encoding a gRNA comprises one nucleotide sequence encoding one or more gRNA.

61. The vector of claim 60, wherein said one nucleotide sequence encoding one or more gRNA is operably linked to a single promoter.

62. The vector of claim 59, wherein said at least one nucleotide sequence encoding a gRNA comprises two or more nucleotide sequences, each nucleotide sequence encoding a gRNA.

63. The vector of claim 62, wherein each nucleotide sequence encoding a gRNA is operably linked to a promoter.

64. The vector of claim 61 or 63, wherein the promoter comprises an RNA polymerase III promoter.

65. The vector of claim 64, wherein said RNA polymerase III promoter comprises a human U6 promoter or a human 7SK promoter.

66. The vector of claim 65, wherein said human U6 promoter comprises a nucleotide sequence having at least 90%, at least 95%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 1672.

67. The vector of claim 65, wherein said human 7SK promoter comprises a nucleotide sequence having at least 90%, at least 95%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 1673.

68. The vector of any one of claims 58-67, wherein the vector further comprises a promoter operably linked to the nucleic acid molecule comprising a polynucleotide encoding an RGN polypeptide.

69. The vector of claim 68, wherein the promoter comprises an RNA polymerase II promoter.

70. The vector of claim 69, wherein said RNA polymerase II promoter comprises a CMV promoter or a CBh promoter.

71. The vector of claim 70, wherein said CMV promoter further comprises a CMV upstream enhancer.

72. The vector of claim 71, wherein said CMV promoter and said CMV upstream enhancer comprises a nucleotide sequence having at least 90%, at least 95%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 1674.

73. The vector of claim 70, wherein said CBh promoter comprises a nucleotide sequence having at least 90%, at least 95%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 1675.

74. The vector of any one of claims 58-73, wherein said vector further comprises a nucleotide sequence encoding a peptide linker.

75. The vector of claim 74, wherein said peptide linker comprises an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 34 or 1928.

76. The vector of any one of claims 58-75, wherein said vector further comprises a polyadenylation (poly A) tail.

77. The vector of claim 76, wherein said polyA tail comprises a SV40 polyA tail.

78. The vector of claim 77, wherein said SV40 polyA tail comprises a nucleotide sequence having at least 90%, at least 95%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 1678.

79. The vector of any one of claims 59-78, wherein the gRNA comprises at least one CRISPR RNA (crRNA) backbone (bb) and at least one spacer.

80. The vector of claim 79, wherein the gRNA comprises two crRNA bb and one spacer in a format of, from 5’ to 3’, crRNA bb-spacer-crRNA bb.

81. The vector of claim 79, wherein the gRNA comprises three crRNA bb, a first spacer, and a second spacer in a format of, from 5’ to 3’, crRNA bb-first spacer-crRNA bb-second spacer- crRNA bb.

82. The vector of claim 79, wherein the gRNA comprises one crRNA bb and one spacer in a format of, from 5’ to 3’, crRNA bb-spacer.

83. The vector of any one of claims 59-82, wherein the gRNA is selected from the group consisting of: a) a gRNA comprising a CRISPR RNA comprising a CRISPR RNA (crRNA) backbone comprising a nucleotide sequence set forth as SEQ ID NO: 5 or that differs from SEQ ID NO: 5 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1; b) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising a nucleotide sequence set forth as SEQ ID NO: 6 or that differs from SEQ ID NO: 6 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2; c) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising a nucleotide sequence set forth as SEQ ID NO: 7 or that differs from SEQ ID NO: 7 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 3; and d) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising a nucleotide sequence set forth as SEQ ID NO: 8 or that differs from SEQ ID NO: 8 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4;(e) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1617, or that differs from SEQ ID NO: 1617 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 4;(f) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1618, or that differs from SEQ ID NO: 1618 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 4;(g) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1619, or that differs from SEQ ID NO: 1619 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 4;(h) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1620, or that differs from SEQ ID NO: 1620 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 4;(i) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1631, or that differs from SEQ ID NO: 1631 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1;(j) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1957, or that differs from SEQ ID NO: 1957 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1930;(k) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1958, or that differs from SEQ ID NO: 1958 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1931;(l) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1959, or that differs from SEQ ID NO: 1959 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1932;(m) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1960, or that differs from SEQ ID NO: 1960 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1933;(n) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1961, or that differs from SEQ ID NO: 1961 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1934;(o) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1962, or that differs from SEQ ID NO: 1962 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1935;(p) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1963, or that differs from SEQ ID NO: 1963 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1936;(q) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1964, or that differs from SEQ ID NO: 1964 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1937;(r) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1965, or that differs from SEQ ID NO: 1965 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1938;(s) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1966, or that differs from SEQ ID NO: 1966 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1939;(t) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1967, or that differs from SEQ ID NO: 1967 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1940;(u) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1968, or that differs from SEQ ID NO: 1968 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1941;(v) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1969, or that differs from SEQ ID NO: 1969 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1942;(w) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1970, or that differs from SEQ ID NO: 1970 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1943;(x) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1971, or that differs from SEQ ID NO: 1971 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1944;(y) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1972, or that differs from SEQ ID NO: 1972 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1945;(z) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1973, or that differs from SEQ ID NO: 1973 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1946;(aa) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1974, or that differs from SEQ ID NO: 1974 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1947;(bb) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1975, or that differs from SEQ ID NO: 1975 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1948;(cc) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1976, or that differs from SEQ ID NO: 1976 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1949;(dd) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1977, or that differs from SEQ ID NO: 1977 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1950;(ee) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1978, or that differs from SEQ ID NO: 1978 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1951;(ff) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1979, or that differs from SEQ ID NO: 1979 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1952;(gg) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1980, or that differs from SEQ ID NO: 1980 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1953;(hh) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1981, or that differs from SEQ ID NO: 1981 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1954;(ii) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1982, or that differs from SEQ ID NO: 1982 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1955; and(jj) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1983, or that differs from SEQ ID NO: 1983 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1956.

84. The vector of claim 83, wherein the gRNA is selected from the group consisting of: a) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 5, wherein said RGN polypeptide comprises the amino acid sequence set forth as SEQ ID NO: 1; b) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 6, wherein said RGN polypeptide comprises the amino acid sequence set forth as SEQ ID NO: 2; c) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 7, wherein said RGN polypeptide comprises the amino acid sequence set forth as SEQ ID NO: 3; and d) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 8, wherein said RGN polypeptide comprises the amino acid sequence set forth as SEQ ID NO: 4;(e) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1617, wherein said RGN polypeptide comprises the amino acid sequence set forth as SEQ ID NO: 4;(f) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1618, wherein said RGN polypeptide comprises the amino acid sequence set forth as SEQ ID NO: 4;(g) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1619, wherein said RGN polypeptide comprises the amino acid sequence set forth as SEQ ID NO: 4;(h) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1620, wherein said RGN polypeptide comprises the amino acid sequence set forth as SEQ ID NO: 4;(i) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1631, wherein said RGN polypeptide comprises the amino acid sequence set forth as SEQ ID NO: 1;(j) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1957, wherein said RGN polypeptide comprises the amino acid sequence set forth as SEQ ID NO: 1930;(k) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1958, wherein said RGN polypeptide comprises the amino acid sequence set forth as SEQ ID NO: 1931;(l) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1959, wherein said RGN polypeptide comprises the amino acid sequence set forth as SEQ ID NO: 1932;(m) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1960, wherein said RGN polypeptide comprises the amino acid sequence set forth as SEQ ID NO: 1933;(n) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1961, wherein said RGN polypeptide comprises the amino acid sequence set forth as SEQ ID NO: 1934;(o) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1962, wherein said RGN polypeptide comprises the amino acid sequence set forth as SEQ ID NO: 1935;(p) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1963, wherein said RGN polypeptide comprises the amino acid sequence set forth as SEQ ID NO: 1936;(q) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1964, wherein said RGN polypeptide comprises the amino acid sequence set forth as SEQ ID NO: 1937;(r) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1965, wherein said RGN polypeptide comprises the amino acid sequence set forth as SEQ ID NO: 1938;(s) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1966, wherein said RGN polypeptide comprises the amino acid sequence set forth as SEQ ID NO: 1939;(t) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1967, wherein said RGN polypeptide comprises the amino acid sequence set forth as SEQ ID NO: 1940;(u) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1968, wherein said RGN polypeptide comprises the amino acid sequence set forth as SEQ ID NO: 1941;(v) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1969, wherein said RGN polypeptide comprises the amino acid sequence set forth as SEQ ID NO: 1942;(w) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1970, wherein said RGN polypeptide comprises the amino acid sequence set forth as SEQ ID NO: 1943;(x) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1971, wherein said RGN polypeptide comprises the amino acid sequence set forth as SEQ ID NO: 1944;(y) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1972, wherein said RGN polypeptide comprises the amino acid sequence set forth as SEQ ID NO: 1945;(z) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1973, wherein said RGN polypeptide comprises the amino acid sequence set forth as SEQ ID NO: 1946;(aa) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1974, wherein said RGN polypeptide comprises the amino acid sequence set forth as SEQ ID NO: 1947;(bb) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1975, wherein said RGN polypeptide comprises the amino acid sequence set forth as SEQ ID NO: 1948;(cc) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1976, wherein said RGN polypeptide comprises the amino acid sequence set forth as SEQ ID NO: 1949;(dd) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1977, wherein said RGN polypeptide comprises the amino acid sequence set forth as SEQ ID NO: 1950;(ee) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1978, wherein said RGN polypeptide comprises the amino acid sequence set forth as SEQ ID NO: 1951;(ff) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1979, wherein said RGN polypeptide comprises the amino acid sequence set forth as SEQ ID NO: 1952;(gg) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1980, wherein said RGN polypeptide comprises the amino acid sequence set forth as SEQ ID NO: 1953;(hh) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1981, wherein said RGN polypeptide comprises the amino acid sequence set forth as SEQ ID NO: 1954;(ii) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1982, wherein said RGN polypeptide comprises the amino acid sequence set forth as SEQ ID NO: 1955; and(jj) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1983, wherein said RGN polypeptide comprises the amino acid sequence set forth as SEQ ID NO: 1956.

85. The vector of any one of claims 59-84, wherein said gRNA comprises a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 39-182, 1405-1412, 1414-1501, 1556, 1567-1616, 1662-1671, and 2126-2658.

86. The vector of any one of claims 58-85, wherein the vector is an adeno-associated viral (AAV) vector.

87. The vector of claim 86, wherein said vector comprises the nucleotide sequence set forth as any one of SEQ ID NOs: 1679-1683.

88. A cell comprising the nucleic acid molecule of any one of claims 1-57 or the vector of any one of claims 58-87.

89. A method for making an RGN polypeptide comprising culturing the cell of claim 88 under conditions in which the RGN polypeptide is expressed.

90. A method for making an RGN polypeptide comprising: introducing into a cell a heterologous nucleic acid molecule comprising a nucleotide sequence encoding an RNA-guided nuclease (RGN) polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as any one of SEQ ID NOs: 1-4, 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 1087, 1105, 1108, 1116, 1121, 1122, 1123, 1126, 1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173, 1223, 1228, 1304, 1322, 1324, 1328, 1330, 1361, 1400, 1930-1956, 2686, and 2687; and culturing said cell under conditions in which the RGN polypeptide is expressed.

91. The method of claim 90, further comprising purifying said RGN polypeptide.

92. The method of claim 90 or 91, wherein said cell further expresses one or more guide RNAs capable of binding to said RGN polypeptide to form an RGN ribonucleoprotein complex.

93. The method of claim 92, further comprising purifying said RGN ribonucleoprotein complex.

94. An RNA-guided nuclease (RGN) polypeptide, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as any one of SEQ ID NOs: 1-4, 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508,539, 597, 602, 678, 707, 708, 717, 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 1087, 1105, 1108, 1116, 1121, 1122, 1123, 1126, 1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173, 1223, 1228, 1304, 1322, 1324, 1328, 1330, 1361, 1400, 1930-1956, 2686, and 2687.

95. The RGN polypeptide of claim 94, wherein said RGN polypeptide comprises the amino acid sequence set forth as any one of SEQ ID NOs: 1-4, 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 1087, 1105, 1108, 1116, 1121, 1122, 1123, 1126,1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173,1223, 1228, 1304, 1322, 1324, 1328, 1330, 1361, 1400, 1930-1956, 2686, and 2687.

96. The RGN polypeptide of claim 94, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1, and wherein the amino acid residue at one or more of amino acid positions 517, 530, 538, 548, 551, 552, 553, 568, 570, 574, 577, 611, 677, 789, 790, and 801 of said RGN polypeptide differs from the corresponding amino acid residue of SEQ ID NO: 1.

97. The RGN polypeptide of claim 96, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1, and wherein the amino acid residue(s) at one or more of amino acid positions 517, 530, 538, 548, 551, 552, 553, 568, 570, 574, 577, 611, 677, 789, 790, and 801 of said RGN polypeptide is a positively charged amino acid residue.

98. The RGN polypeptide of claim 96 or 97, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1, and wherein the amino acid residue(s) at one or more of amino acid positions 517, 530, 538, 548, 551, 552, 553, 568, 570, 574, 577, 611, 677, 789, 790, and 801 of said RGN polypeptide is an arginine (R).

99. The RGN polypeptide of claim 98, wherein said RGN polypeptide comprises an amino acid sequence having 100% sequence identity to the amino acid sequence selected from:(a) the amino acid sequence set forth as SEQ ID NO: 450;(b) the amino acid sequence set forth as SEQ ID NO: 463;(c) the amino acid sequence set forth as SEQ ID NO: 471;(d) the amino acid sequence set forth as SEQ ID NO: 481;(e) the amino acid sequence set forth as SEQ ID NO: 484;(f) the amino acid sequence set forth as SEQ ID NO: 485;(g) the amino acid sequence set forth as SEQ ID NO: 486;(h) the amino acid sequence set forth as SEQ ID NO: 500;(i) the amino acid sequence set forth as SEQ ID NO: 502;(j) the amino acid sequence set forth as SEQ ID NO: 505;(k) the amino acid sequence set forth as SEQ ID NO: 508;(l) the amino acid sequence set forth as SEQ ID NO: 539;(m) the amino acid sequence set forth as SEQ ID NO: 602;(n) the amino acid sequence set forth as SEQ ID NO: 707;(o) the amino acid sequence set forth as SEQ ID NO: 708; and(p) the amino acid sequence set forth as SEQ ID NO: 717.

100. The RGN polypeptide of claim94, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1, and wherein the amino acid residues are arginines (Rs) at a combination of amino acid positions selected from the following combinations of amino acid positions of said RGN polypeptide:(a) amino acid positions 517, 530, 552, and 677;(b) amino acid positions 530, 538, 552, and 801;(c) amino acid positions 530, 552, 677, and 801;(d) amino acid positions 530, 577, and 790;(e) amino acid positions 517, 530, 538, 552, and 677;(f) amino acid positions 530, 538, 677, and 801;(g) amino acid positions 538, 552, 677, and 801;(h) amino acid positions 517, 530, 538, and 552;(i) amino acid positions 517 and 530;(j) amino acid positions 530, 538, and 552;(k) amino acid positions 517, 530, 538, and 677;(l) amino acid positions 517, 552, 677, and 801;(m) amino acid positions 530, 677, and 801;(n) amino acid positions 517, 530, 677, and 801;(o) amino acid positions 517, 530, and 677;(p) amino acid positions 530, 538, 552, and 677;(q) amino acid positions 517, 552, and 677; and(r) amino acid positions 517, 530, 552, 677, and 801.

101. The RGN polypeptide of claim 100, wherein said RGN polypeptide comprises an amino acid sequence having 100% sequence identity to the amino acid sequence selected from:(a) the amino acid sequence set forth as SEQ ID NO: 1072;(b) the amino acid sequence set forth as SEQ ID NO: 1080;(c) the amino acid sequence set forth as SEQ ID NO: 1082;(d) the amino acid sequence set forth as SEQ ID NO: 1105;(e) the amino acid sequence set forth as SEQ ID NO: 1084;(f) the amino acid sequence set forth as SEQ ID NO: 1081;(g) the amino acid sequence set forth as SEQ ID NO: 1083;(h) the amino acid sequence set forth as SEQ ID NO: 1069;(i) the amino acid sequence set forth as SEQ ID NO: 1034;(j) the amino acid sequence set forth as SEQ ID NO: 1059;(k) the amino acid sequence set forth as SEQ ID NO: 1070;(l) the amino acid sequence set forth as SEQ ID NO: 1078;(m) the amino acid sequence set forth as SEQ ID NO: 1064;(n) the amino acid sequence set forth as SEQ ID NO: 1074;(o) the amino acid sequence set forth as SEQ ID NO: 1051;(p) the amino acid sequence set forth as SEQ ID NO: 1079;(q) the amino acid sequence set forth as SEQ ID NO: 1056; and(r) the amino acid sequence set forth as SEQ ID NO: 1087.

102. The RGN polypeptide of claim 94, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4, and wherein the amino acid residue(s) at one or more of amino acid positions 377, 378, 438, 625, 638, 641, 645, 691, 692, 695, 698, 702, 707, 747, and 755 of said RGN polypeptide differs from the corresponding amino acid residue of SEQ ID NO: 4.

103. The RGN polypeptide of claim 102, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4, and wherein the amino acid residue(s) at one or more of amino acid positions 377, 378, 438, 625, 638, 641, 645, 691, 692, 695, 698, 702, 707, 747, and 755 of said RGN polypeptide is a positively charged amino acid residue.

104. The RGN polypeptide of claim 102 or 103, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4, and wherein the amino acid residue(s) at one or more of amino acid positions 377, 378, 438, 625, 638, 641, 645, 691, 692, 695, 698, 702, 707, 747, and 755 of said RGN polypeptide is an R.

105. The RGN polypeptide of claim 104, wherein said RGN polypeptide comprises an amino acid sequence having 100% sequence identity to the amino acid sequence selected from:(a) the amino acid sequence set forth as SEQ ID NO: 809;(b) the amino acid sequence set forth as SEQ ID NO: 810;(c) the amino acid sequence set forth as SEQ ID NO: 866;(d) the amino acid sequence set forth as SEQ ID NO: 898;(e) the amino acid sequence set forth as SEQ ID NO: 909;(f) the amino acid sequence set forth as SEQ ID NO: 912;(g) the amino acid sequence set forth as SEQ ID NO: 916;(h) the amino acid sequence set forth as SEQ ID NO: 960;(i) the amino acid sequence set forth as SEQ ID NO: 961;(j) the amino acid sequence set forth as SEQ ID NO: 963;(k) the amino acid sequence set forth as SEQ ID NO: 966;(l) the amino acid sequence set forth as SEQ ID NO: 970;(m) the amino acid sequence set forth as SEQ ID NO: 975;(n) the amino acid sequence set forth as SEQ ID NO: 1012; and(o) the amino acid sequence set forth as SEQ ID NO: 1019.

106. The RGN polypeptide of claim 94, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4, and wherein the amino acid residues are arginines (Rs) at a combination of amino acid positions selected from the following combinations of amino acid positions of said RGN polypeptide:(a) amino acid positions 377, 625, and 698;(b) amino acid positions 377, 625, 638, 698, and 747;(c) amino acid positions 377, 625, 691, 698, and 747;(d) amino acid positions 377, 638, 691, and 747;(e) amino acid positions 377, 638, 691, 698, and 747;(f) amino acid positions 377, 625, and 691;(g) amino acid positions 377, 625, 698, and 747;(h) amino acid positions 377, 638, 691, and 698;(i) amino acid positions 377, 625, and 638;(j) amino acid positions 377, 638, 698, and 747;(k) amino acid positions 377, 625, 638, 691, and 698;(l) amino acid positions 625, 638, and 698;(m) amino acid positions 377, 625, 691, and 698;(n) amino acid positions 377, 638, and 698;(o) amino acid positions 625, 691, 698, and 747;(p) amino acid positions 638 and 698; and(q) amino acid positions 377 and 691.

107. The RGN polypeptide of claim 106, wherein said RGN polypeptide comprises an amino acid sequence having 100% sequence identity to the amino acid sequence selected from:(a) the amino acid sequence set forth as SEQ ID NO: 1123;(b) the amino acid sequence set forth as SEQ ID NO: 1158;(c) the amino acid sequence set forth as SEQ ID NO: 1159;(d) the amino acid sequence set forth as SEQ ID NO: 1148;(e) the amino acid sequence set forth as SEQ ID NO: 1160;(f) the amino acid sequence set forth as SEQ ID NO: 1122;(g) the amino acid sequence set forth as SEQ ID NO: 1146;(h) the amino acid sequence set forth as SEQ ID NO: 1147;(i) the amino acid sequence set forth as SEQ ID NO: 1121;(j) the amino acid sequence set forth as SEQ ID NO: 1149;(k) the amino acid sequence set forth as SEQ ID NO: 1156;(l) the amino acid sequence set forth as SEQ ID NO: 1132;(m) the amino acid sequence set forth as SEQ ID NO: 1144;(n) the amino acid sequence set forth as SEQ ID NO: 1126;(o) the amino acid sequence set forth as SEQ ID NO: 1154;(p) the amino acid sequence set forth as SEQ ID NO: 1116; and(q) the amino acid sequence set forth as SEQ ID NO: 1108.

108. The RGN polypeptide of claim 94, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4, and wherein the amino acid residue(s) at one or more of amino acid positions 685, 691, 692, 695, 698, 702, 707, 736, 740, 743, 747, 751, 752, 753, 755, 757, 758, 760, 764, and 766 of said RGN polypeptide differs from the corresponding amino acid residue of SEQ ID NO: 4.

109. The RGN polypeptide of claim 108, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4, and wherein the amino acid residue(s) at one or more of amino acid positions 685, 691, 692, 695, 698, 702, 707, 736, 740, 743, 747, 751, 752, 753, 755, 757, 758, 760, 764, and 766 of said RGN polypeptide is a positively charged amino acid residue.

110. The RGN polypeptide of claim 108 or 109, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4, and wherein the amino acid residue(s) at one or more of amino acid positions 685, 691, 692, 695, 698, 702, 707, 736, 740, 743, 747, 751, 752, 753, 755, 757, 758, 760, 764, and 766 of said RGN polypeptide is an R or a lysine (K).

111. The RGN polypeptide of claim 94, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4, and wherein the amino acid residues are arginines (Rs) and / or lysines (Ks) at a combination of amino acid positions selected from the following combinations of amino acid positions of said RGN polypeptide:(a) amino acid positions 736, 743, 752, 753, 755, 757, 758, and 760;(b) amino acid positions 740, 747, 751, 753, 755, 760, 764, and 766;(c) amino acid positions 685, 691, and 698; and(d) amino acid positions 685, 692, 695, 702, and 707.

112. The RGN polypeptide of claim 111, wherein said RGN polypeptide comprises an amino acid sequence having 100% sequence identity to the amino acid sequence selected from:(a) the amino acid sequence set forth as SEQ ID NO: 1168;(b) the amino acid sequence set forth as SEQ ID NO: 1171;(c) the amino acid sequence set forth as SEQ ID NO: 1169; and(d) the amino acid sequence set forth as SEQ ID NO: 1170.

113. The RGN polypeptide of claim 94, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4, and wherein said RGN polypeptide comprises a deletion of amino acid residues corresponding to amino acid residues 751-769 of SEQ ID NO: 4.

114. The RGN polypeptide of claim 113, wherein said RGN polypeptide comprises the amino acid sequence set forth as SEQ ID NO: 1173.

115. The RGN polypeptide of claim 94, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1, and wherein one or more of amino acid positions 293, 294, 359, 360, 361, 401, 402, 403, 596, and 862 of said RGN polypeptide comprises substitution or insertion of one or more glycines (G) immediately N-terminal to, at, or immediately C-terminal to said amino acid position.

116. The RGN polypeptide of claim 115, wherein said substitution or insertion of one or more G comprises substitution or insertion of one, two, or three G.

117. The RGN polypeptide of claim 115, wherein said substitution or insertion of one or more G is selected from:(a) an insertion of three G between amino acid positions 293 and 294;(b) an insertion of two G between amino acid positions 359 and 360;(c) a substitution of G at amino acid position 361;(d) an insertion of one G between amino acid positions 402 and 403;(e) a substitution of G at amino acid position 401 ;(f) a substitution of G at amino acid position 596; and(g) a substitution of G at amino acid position 862.

118. The RGN polypeptide of claim 117, wherein said RGN polypeptide comprises an amino acid sequence having 100% sequence identity to the amino acid sequence selected from:(a) the amino acid sequence set forth as SEQ ID NO: 1304;(b) the amino acid sequence set forth as SEQ ID NO: 1322;(c) the amino acid sequence set forth as SEQ ID NO: 1324;(d) the amino acid sequence set forth as SEQ ID NO: 1328;(e) the amino acid sequence set forth as SEQ ID NO: 1330;(f) the amino acid sequence set forth as SEQ ID NO: 1361; and(g) the amino acid sequence set forth as SEQ ID NO: 1400.

119. The RGN polypeptide of claim 94, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4, and wherein one or more of amino acid positions 566, 567, and 584 of said RGN polypeptide comprises substitution or insertion of one or more G immediately N-terminal to, at, or immediately C-terminal to said amino acid position.

120. The RGN polypeptide of claim 119, wherein said substitution or insertion of one or more G comprises substitution or insertion of one, two, or three G.

121. The RGN polypeptide of claim 119 or 120, wherein said substitution or insertion of one or more G is selected from: (a) a substitution of G at amino acid position 566 and a substitution of G at amino acid position 567; and (b) a substitution of G at amino acid position 584.

122. The RGN polypeptide of claim 121, wherein said RGN polypeptide comprises the amino acid sequence selected from: (a) the amino acid sequence set forth as SEQ ID NO: 1223; and (b) the amino acid sequence set forth as SEQ ID NO: 1228.

123. The RGN polypeptide of any one of claims 94-122, wherein said RGN polypeptide is operably fused to a heterologous polypeptide.

124. The RGN polypeptide of claim 123, wherein the heterologous polypeptide comprises a base-editing polypeptide, a polymerase editing polypeptide, an effector domain, an expression modulator, a detectable label, or a purification tag.

125. The RGN polypeptide of claim 124, wherein the base editing polypeptide comprises a deaminase.

126. The RGN polypeptide of claim 124, wherein the polymerase editing polypeptide comprises a DNA polymerase.

127. The RGN polypeptide of claim 124, wherein the polymerase editing polypeptide comprises a reverse transcriptase.

128. The RGN polypeptide of claim 124, wherein the heterologous polypeptide comprises an effector domain.

129. The RGN polypeptide of claim 128, wherein the effector domain is operably fused at the N-terminus or the C-terminus of the RGN polypeptide.

130. The RGN polypeptide of claim 128, wherein the effector domain is operably fused at an internal location of the RGN polypeptide.

131. The RGN polypeptide of any one of claims 128-130, wherein the effector domain comprises a cleavage domain, a deaminase domain, or an expression modulator domain.

132. The RGN polypeptide of claim 131, wherein the expression modulator domain comprises a trancriptional activation domain, a transcriptional repressor domain, or an epigenetic modification domain.

133. The RGN polypeptide of any one of claims 94-132, wherein said RGN polypeptide is capable of binding a target sequence in a target nucleic acid molecule in an RNA-guided sequence specific manner when bound to a guide RNA (gRNA), wherein said target sequence comprises a target strand and a non-target strand, and wherein said gRNA is capable of hybridizing to the target strand of the target sequence.

134. The RGN polypeptide of claim 133, wherein said target sequence is located adjacent to and 3' of a protospacer adjacent motif (PAM).

135. The RGN polypeptide of claim 134, wherein a) the RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO: 1 recognizes the PAM having a consensus nucleotide sequence set forth as AYG, wherein the PAM is adjacent to and 5' of the target sequence; b) the RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO: 2, 1933, or 1944, recognizes the PAM having a consensus nucleotide sequence set forth as ATTN, wherein the PAM is adjacent to and 5' of the target sequence; c) the RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO: 3, 1938, 1943, 1945, 1947, or 1949, recognizes the PAM having a consensus nucleotide sequence set forth as VTTN, wherein the PAM is adjacent to and 5' of the target sequence; d) the RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO: 4, 1930, 1932, 1934, 1936, 1939, 1940, 1941, 1942, 1946, 1948, 1952, 1954, 1955, or 1956, recognizes the PAM having a consensus nucleotide sequence set forth as TTN, wherein the PAM is adjacent to and 5' of the target sequence; e) the RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO: 1931 recognizes the PAM having a consensus nucleotide sequence set forth as STTN, wherein the PAM is adjacent to and 5' of the target sequence; f) the RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO: 1935 recognizes the PAM having a consensus nucleotide sequence set forth as TTH, wherein the PAM is adjacent to and 5' of the target sequence; g) the RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO: 1937 or 1950 recognizes the PAM having a consensus nucleotide sequence set forth as RTTN, wherein the PAM is adjacent to and 5' of the target sequence; h) the RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO: 1951 recognizes the PAM having a consensus nucleotide sequence set forth as RTYN, wherein the PAM is adjacent to and 5' of the target sequence; andi) the RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO: 1953 recognizes the PAM having a consensus nucleotide sequence set forth as ATG, wherein the PAM is adjacent to and 5' of the target sequence.

136. The RGN polypeptide of any one of claims 133-135, wherein said RGN polypeptide is capable of cleaving said target nucleic acid molecule upon binding.

137. The RGN polypeptide of claim 136, wherein cleavage by said RGN polypeptide generates a double-stranded break.

138. The RGN polypeptide of claim 94, wherein said RGN polypeptide is nuclease inactive.

139. The RGN polypeptide of claim 138, wherein said RGN polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to any one of SEQ ID NOs: 408, 597, 678, 827, 896, 979, 2686, and 2687.

140. The RGN polypeptide of any one of claims 94-139, wherein the RGN polypeptide is operably fused to a base -editing polypeptide.

141. The RGN polypeptide of claim 140, wherein the base-editing polypeptide comprises a deaminase.

142. The RGN polypeptide of any one of claims 94-139, wherein the RGN polypeptide is operably fused to a polymerase editing polypeptide.

143. The RGN polypeptide of claim 142, wherein said polymerase editing polypeptide comprises a DNA polymerase.

144. The RGN polypeptide of claim 142, wherein said polymerase editing polypeptide comprises a reverse transcriptase.

145. The RGN polypeptide of any one of claims 94-144, wherein the RGN polypeptide comprises one or more nuclear localization signals (NLS).

146. The RGN polypeptide of claim 145, wherein the one or more NLS comprises an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to any one of SEQ ID NOs: SEQ ID NOs: 33, 35, 407, and 1927.

147. A ribonucleoprotein (RNP) complex comprising the RGN polypeptide of any one of claims 94-146 and a guide RNA bound to the RGN polypeptide.

148. A guide RNA (gRNA) comprising a CRISPR RNA (crRNA), wherein said crRNA comprises a crRNA backbone and a spacer, wherein said crRNA backbone is selected from the group consisting of: a) a crRNA backbone comprising a nucleotide sequence set forth as SEQ ID NO: 5 or that differs from SEQ ID NO: 5 by 1 to 5 nucleotides; b) a crRNA backbone comprising a nucleotide sequence set forth as SEQ ID NO: 6 or that differs from SEQ ID NO: 6 by 1 to 5 nucleotides;c) a crRNA backbone comprising a nucleotide sequence set forth as SEQ ID NO: 7 or that differs from SEQ ID NO: 7 by 1 to 5 nucleotides; d) a crRNA backbone comprising a nucleotide sequence set forth as SEQ ID NO: 8 or that differs from SEQ ID NO: 8 by 1 to 5 nucleotides;(e) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO:1617, or that differs from SEQ ID NO: 1617 by 1 to 5 nucleotides;(f) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO:1618, or that differs from SEQ ID NO: 1618 by 1 to 5 nucleotides;(g) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO:1619, or that differs from SEQ ID NO: 1619 by 1 to 5 nucleotides;(h) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO:1620, or that differs from SEQ ID NO: 1620 by 1 to 5 nucleotides;(i) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1631, or that differs from SEQ ID NO: 1631 by 1 to 5 nucleotides;(j) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO:1957, or that differs from SEQ ID NO: 1957 by 1 to 5 nucleotides;(k) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO:1958, or that differs from SEQ ID NO: 1958 by 1 to 5 nucleotides;(l) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO:1959, or that differs from SEQ ID NO: 1959 by 1 to 5 nucleotides;(m) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO:1960, or that differs from SEQ ID NO: 1960 by 1 to 5 nucleotides;(n) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO:1961, or that differs from SEQ ID NO: 1961 by 1 to 5 nucleotides;(o) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO:1962, or that differs from SEQ ID NO: 1962 by 1 to 5 nucleotides;(p) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO:1963, or that differs from SEQ ID NO: 1963 by 1 to 5 nucleotides;(q) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO:1964, or that differs from SEQ ID NO: 1964 by 1 to 5 nucleotides;(r) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO:1965, or that differs from SEQ ID NO: 1965 by 1 to 5 nucleotides;(s) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO:1966, or that differs from SEQ ID NO: 1966 by 1 to 5 nucleotides;(t) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO:1967, or that differs from SEQ ID NO: 1967 by 1 to 5 nucleotides;(u) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO:1968, or that differs from SEQ ID NO: 1968 by 1 to 5 nucleotides;(v) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO:1969, or that differs from SEQ ID NO: 1969 by 1 to 5 nucleotides;(w) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO:1970, or that differs from SEQ ID NO: 1970 by 1 to 5 nucleotides;(x) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO:1971, or that differs from SEQ ID NO: 1971 by 1 to 5 nucleotides;(y) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO:1972, or that differs from SEQ ID NO: 1972 by 1 to 5 nucleotides;(z) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO:1973, or that differs from SEQ ID NO: 1973 by 1 to 5 nucleotides;(aa) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO:1974, or that differs from SEQ ID NO: 1974 by 1 to 5 nucleotides;(bb) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO:1975, or that differs from SEQ ID NO: 1975 by 1 to 5 nucleotides;(cc) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO:1976, or that differs from SEQ ID NO: 1976 by 1 to 5 nucleotides;(dd) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO:1977, or that differs from SEQ ID NO: 1977 by 1 to 5 nucleotides;(ee) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO:1978, or that differs from SEQ ID NO: 1978 by 1 to 5 nucleotides;(ff) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO:1979, or that differs from SEQ ID NO: 1979 by 1 to 5 nucleotides;(gg) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO:1980, or that differs from SEQ ID NO: 1980 by 1 to 5 nucleotides;(hh) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO:1981, or that differs from SEQ ID NO: 1981 by 1 to 5 nucleotides;(ii) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO:1982, or that differs from SEQ ID NO: 1982 by 1 to 5 nucleotides; and(jj) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1983, or that differs from SEQ ID NO: 1983 by 1 to 5 nucleotides.

149. The gRNA of claim 148, wherein said crRNA backbone is selected from the group consisting of: a) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 5; b) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 6;c) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 7; d) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 8;(e) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO:1617;(f) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1618;(g) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1619;(h) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1620;(i) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1631;(j) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1957;(k) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1958;(l) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1959;(m) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1960;(n) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1961;(o) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1962;(p) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1963;(q) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1964;(r) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1965;(s) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1966;(t) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1967;(u) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO:1968;(v) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1969;(w) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1970;(x) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1971;(y) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1972;(z) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1973;(aa) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1974;(bb) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1975;(cc) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1976;(dd) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1977;(ee) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1978;(ff) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1979;(gg) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1980;(hh) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1981;(ii) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1982; and(jj) a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1983.

150. The gRNA of claim 148 or 149, wherein the gRNA does not comprise a tracrRNA.

151. The gRNA of claim 148 or 149, wherein the gRNA consists of the cRNA.

152. The gRNA of any one of claims 148-151, wherein the gRNA comprises a total length of 35-250 nucleotides (nt), or 35-170 nt, or 35-125 nt, or 40-100 nt, or 40-70 nt, or 40-60 nt.

153. The gRNA of claim any one of claims 148-151, wherein the gRNA comprises a crRNA backbone comprising a total length of at most 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, 26 nt, 27 nt, 28 nt, 29 nt, 30 nt, 31 nt, 32 nt, 33 nt, 34 nt, 35 nt, 36 nt, 37 nt, 38 nt, 39 nt, or 40 nt.

154. The gRNA of any one of claims 148-153, wherein said spacer comprises the nucleotide sequence set forth as any one of SEQ ID NOs: 294-396, 1789-1796, 1798-1880, 1890- 1893, and 2082-2125.

155. The gRNA of any one of claims 148-154, wherein said crRNA comprises the nucleotide sequence set forth as any one of SEQ ID NOs: 39-182, 1405-1412, 1414-1501, 1556, 1567-1616, 1662-1671, and 2126-2658.

156. The gRNA of any one of claims 148-155, wherein said gRNA is capable of hybridizing to the target strand of a target sequence in a target nucleic acid molecule in a sequence specific manner through the spacer of said crRNA when said gRNA is bound to an RNA-guided nuclease (RGN) polypeptide.

157. The gRNA of claim 156, wherein said RGN polypeptide is selected from the group consisting of: a) a RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 5 or that differs from SEQ ID NO: 5 by 1 to 5 nucleotides; b) a RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 6 or that differs from SEQ ID NO: 6 by 1 to 5 nucleotides; c) a RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 3, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 7 or that differs from SEQ ID NO: 7 by 1 to 5 nucleotides; d) a RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4, wherein said crRNA backbone comprises a nucleotide sequence set forth as SEQ ID NO: 8 or that differs from SEQ ID NO: 8 by 1 to 5 nucleotides;(e) a RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 4, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 1617, or that differs from SEQ ID NO: 1617 by 1 to 5 nucleotides;(f) a RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 4, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 1618, or that differs from SEQ ID NO: 1618 by 1 to 5 nucleotides;(g) a RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 4, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 1619, or that differs from SEQ ID NO: 1619 by 1 to 5 nucleotides;(h) a RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 4, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 1620, or that differs from SEQ ID NO: 1620 by 1 to 5 nucleotides;(i) a RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 1631, or that differs from SEQ ID NO: 1631 by 1 to 5 nucleotides;(j) a RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1930, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 1957, or that differs from SEQ ID NO: 1957 by 1 to 5 nucleotides;(k) a RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1931, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 1958, or that differs from SEQ ID NO: 1958 by 1 to 5 nucleotides;(l) a RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1932, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 1959, or that differs from SEQ ID NO: 1959 by 1 to 5 nucleotides;(m) a RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1933, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 1960, or that differs from SEQ ID NO: 1960 by 1 to 5 nucleotides;(n) a RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1934, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 1961, or that differs from SEQ ID NO: 1961 by 1 to 5 nucleotides;(o) a RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1935, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 1962, or that differs from SEQ ID NO: 1962 by 1 to 5 nucleotides;(p) a RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1936, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 1963, or that differs from SEQ ID NO: 1963 by 1 to 5 nucleotides;(q) a RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1937, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 1964, or that differs from SEQ ID NO: 1964 by 1 to 5 nucleotides;(r) a RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1938, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 1965, or that differs from SEQ ID NO: 1965 by 1 to 5 nucleotides;(s) a RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1939, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 1966, or that differs from SEQ ID NO: 1966 by 1 to 5 nucleotides;(t) a RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1940, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 1967, or that differs from SEQ ID NO: 1967 by 1 to 5 nucleotides;(u) a RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1941, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 1968, or that differs from SEQ ID NO: 1968 by 1 to 5 nucleotides;(v) a RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1942, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 1969, or that differs from SEQ ID NO: 1969 by 1 to 5 nucleotides;(w) a RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1943, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 1970, or that differs from SEQ ID NO: 1970 by 1 to 5 nucleotides;(x) a RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1944, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 1971, or that differs from SEQ ID NO: 1971 by 1 to 5 nucleotides;(y) a RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1945, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 1972, or that differs from SEQ ID NO: 1972 by 1 to 5 nucleotides;(z) a RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1946, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 1973, or that differs from SEQ ID NO: 1973 by 1 to 5 nucleotides;(aa) a RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1947, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 1974, or that differs from SEQ ID NO: 1974 by 1 to 5 nucleotides;(bb) a RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1948, wherein said crRNA backbone comprisesthe nucleotide sequence set forth as SEQ ID NO: 1975, or that differs from SEQ ID NO: 1975 by 1 to 5 nucleotides;(cc) a RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1949, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 1976, or that differs from SEQ ID NO: 1976 by 1 to 5 nucleotides;(dd) a RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1950, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 1977, or that differs from SEQ ID NO: 1977 by 1 to 5 nucleotides;(ee) a RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1951, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 1978, or that differs from SEQ ID NO: 1978 by 1 to 5 nucleotides;(ff) a RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1952, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 1979, or that differs from SEQ ID NO: 1979 by 1 to 5 nucleotides;(gg) a RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1953, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 1980, or that differs from SEQ ID NO: 1980 by 1 to 5 nucleotides;(hh) a RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1954, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 1981, or that differs from SEQ ID NO: 1981 by 1 to 5 nucleotides;(ii) a RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1955, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 1982, or that differs from SEQ ID NO: 1982 by 1 to 5 nucleotides; and(jj) a RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1956, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 1983, or that differs from SEQ ID NO: 1983 by 1 to 5 nucleotides.

158. The gRNA of claim 156 or 157, wherein said RGN polypeptide is selected from the group consisting of: a) a RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO: 1, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 5; b) a RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO: 2, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 6; c) a RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO: 3, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 7; d) a RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO: 4, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 8;(e) a RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO: 4, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 1617;(f) a RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO: 4, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 1618;(g) a RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO: 4, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 1619(h) a RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO: 4, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 1620;(i) a RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO: 1, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 1631;(j) a RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO:1930, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 1957;(k) a RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO:1931, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 1958;(l) a RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO:1932, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 1959;(m) a RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO:1933, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 1960;(n) a RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO:1934, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 1961;(o) a RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO:1935, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 1962;(p) a RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO:1936, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 1963;(q) a RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO:1937, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 1964;(r) a RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO:1938, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 1965;(s) a RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO:1939, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 1966;(t) a RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO:1940, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 1967;(u) a RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO:1941, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 1968;(v) a RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO:1942, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 1969;(w) a RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO:1943, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 1970;(x) a RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO:1944, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 1971;(y) a RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO:1945, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 1972;(z) a RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO:1946, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 1973;(aa) a RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO:1947, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 1974;(bb) a RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO:1948, wherein said crRNA backbone comprisesthe nucleotide sequence set forth as SEQ ID NO: 1975;(cc) a RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO:1949, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 1976;(dd) a RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO:1950, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 1977;(ee) a RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO:1951, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 1978;(ff) a RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO:1952, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 1979;(gg) a RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO:1953, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 1980;(hh) a RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO:1954, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 1981;(ii) a RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO:1955, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 1982; and(jj) a RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO:1956, wherein said crRNA backbone comprises the nucleotide sequence set forth as SEQ ID NO: 1983.

159. The gRNA of any one of claims 156-158, wherein said target sequence is located adjacent to and 3' of a protospacer adjacent motif (PAM).

160. The gRNA of claim 159, wherein a) the RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO: 1 recognizes the PAM having a consensus nucleotide sequence set forth as AYG, wherein the PAM is adjacent to and 5' of the target sequence; b) the RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO: 2, 1933, or 1944, recognizes the PAM having a consensus nucleotide sequence set forth as ATTN, wherein the PAM is adjacent to and 5' of the target sequence; c) the RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO: 3, 1938, 1943, 1945, 1947, or 1949, recognizes the PAM having a consensus nucleotide sequence set forth as VTTN, wherein the PAM is adjacent to and 5' of the target sequence; and d) the RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO: 4, 1930, 1932, 1934, 1936, 1939, 1940, 1941, 1942, 1946, 1948, 1952, 1954, 1955, or 1956, recognizes the PAM having a consensus nucleotide sequence set forth as TTN, wherein the PAM is adjacent to and 5' of the target sequence; e) the RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO: 1931 recognizes the PAM having a consensus nucleotide sequence set forth as STTN, wherein the PAM is adjacent to and 5' of the target sequence; f) the RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO: 1935 recognizes the PAM having a consensus nucleotide sequence set forth as TTH, wherein the PAM is adjacent to and 5' of the target sequence; g) the RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO: 1937 or 1950 recognizes the PAM having a consensus nucleotide sequence set forth as RTTN, wherein the PAM is adjacent to and 5' of the target sequence;h) the RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO: 1951 recognizes the PAM having a consensus nucleotide sequence set forth as RTYN, wherein the PAM is adjacent to and 5' of the target sequence; and i) the RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO: 1953 recognizes the PAM having a consensus nucleotide sequence set forth as ATG, wherein the PAM is adjacent to and 5' of the target sequence.

161. A nucleic acid molecule comprising at least one polynucleotide encoding the gRNA of any one of claims 148-160.

162. A vector comprising the nucleic acid molecule of claim 161.

163. A cell comprising the RGN polypeptide of any one of claims 94-146, the RNP complex of claim 147, the gRNA of any one of claims 148-160, the nucleic acid molecule of claim 161, or the vector of claim 162.

164. The cell of claim 163, wherein the cell is a eukaryotic cell.

165. An RNA-guided nuclease (RGN) system for binding one or more target sequence, said system comprising: a) one or more guide RNA (gRNA), or one or more polynucleotide comprising one or more nucleotide sequence encoding the one or more gRNA, wherein the one or more gRNA comprises a CRISPR RNA (crRNA) comprising a crRNA backbone and a spacer; and b) an RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as any one of SEQ ID NOs: 1-4, 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 809, 810, 827, 866, 896, 898,909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1034, 1051, 1056, 1059, 1064, 1069,1070, 1072, 1074, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 1087, 1105, 1108, 1116, 1121, 1122,1123, 1126, 1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170,1171, 1173, 1223, 1228, 1304, 1322, 1324, 1328, 1330, 1361, 1400, 1930-1956, 2686, and 2687, or a polynucleotide comprising a nucleotide sequence encoding the RGN polypeptide.

166. The RGN system of claim 165, wherein said RGN polypeptide comprises the amino acid sequence set forth as any one of SEQ ID NOs: 1-4, 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 1087, 1105, 1108, 1116, 1121, 1122, 1123, 1126, 1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173, 1223, 1228, 1304, 1322, 1324, 1328, 1330, 1361, 1400, 1930-1956, 2686, and 2687.

167. The RGN system of claim 165 or 166, wherein each of the one or more gRNA is capable of hybridizing to the target strand of a corresponding target sequence and forming a complex with the RGN polypeptide to direct said RGN polypeptide to bind to said target sequence.

168. The RGN system of any one of claims 165-167, wherein at least one of said one or more nucleotide sequence encoding the one or more gRNA and said nucleotide sequence encoding the RGN polypeptide is operably linked to a promoter heterologous to said nucleotide sequence.

169. The RGN system of any one of claims 165-168, wherein said one or more nucleotide sequence encoding the one or more gRNA comprises:(i) one nucleotide sequence encoding one or more gRNA; or(ii) two or more nucleotide sequences, each nucleotide sequence encoding a gRNA.

170. The RGN system of claim 169, wherein said one nucleotide sequence encoding one or more gRNA of (i) is operably linked to a single promoter.

171. The RGN system of claim 169, wherein each nucleotide sequence encoding a gRNA of (ii) is operably linked to a promoter.

172. The RGN system of any one of claims 165-167, wherein said polynucleotide comprising a nucleotide sequence encoding the RGN polypeptide is an mRNA comprising a heterologous 5 ’ untranslated region (UTR) and / or a heterologous 3 ’ UTR.

173. The RGN system of any one of claims 165-172, wherein said one or more gRNA is selected from the group consisting of: a) a gRNA comprising a crRNA backbone comprising a nucleotide sequence set forth as SEQ ID NO: 5 or that differs from SEQ ID NO: 5 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1; b) a gRNA comprising a crRNA backbone comprising a nucleotide sequence set forth as SEQ ID NO: 6 or that differs from SEQ ID NO: 6 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2; c) a gRNA comprising a crRNA backbone comprising a nucleotide sequence set forth as SEQ ID NO: 7 or that differs from SEQ ID NO: 7 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 3; d) a gRNA comprising a crRNA backbone comprising a nucleotide sequence set forth as SEQ ID NO: 8 or that differs from SEQ ID NO: 8 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4;(e) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1617, or that differs from SEQ ID NO: 1617 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 4;(f) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1618, or that differs from SEQ ID NO: 1618 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 4;(g) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1619, or that differs from SEQ ID NO: 1619 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 4;(h) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1620, or that differs from SEQ ID NO: 1620 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 4;(i) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1631, or that differs from SEQ ID NO: 1631 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1;(j) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1957, or that differs from SEQ ID NO: 1957 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1930;(k) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1958, or that differs from SEQ ID NO: 1958 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1931;(l) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1959, or that differs from SEQ ID NO: 1959 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1932;(m) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1960, or that differs from SEQ ID NO: 1960 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1933;(n) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1961, or that differs from SEQ ID NO: 1961 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1934;(o) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1962, or that differs from SEQ ID NO: 1962 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1935;(p) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1963, or that differs from SEQ ID NO: 1963 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1936;(q) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1964, or that differs from SEQ ID NO: 1964 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1937;(r) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1965, or that differs from SEQ ID NO: 1965 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1938;(s) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1966, or that differs from SEQ ID NO: 1966 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1939;(t) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1967, or that differs from SEQ ID NO: 1967 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1940;(u) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1968, or that differs from SEQ ID NO: 1968 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1941;(v) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1969, or that differs from SEQ ID NO: 1969 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1942;(w) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1970, or that differs from SEQ ID NO: 1970 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1943;(x) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1971, or that differs from SEQ ID NO: 1971 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1944;(y) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1972, or that differs from SEQ ID NO: 1972 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1945;(z) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1973, or that differs from SEQ ID NO: 1973 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1946;(aa) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1974, or that differs from SEQ ID NO: 1974 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1947;(bb) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1975, or that differs from SEQ ID NO: 1975 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1948;(cc) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1976, or that differs from SEQ ID NO: 1976 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1949;(dd) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1977, or that differs from SEQ ID NO: 1977 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1950;(ee) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1978, or that differs from SEQ ID NO: 1978 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1951;(ff) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1979, or that differs from SEQ ID NO: 1979 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1952;(gg) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1980, or that differs from SEQ ID NO: 1980 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1953;(hh) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1981, or that differs from SEQ ID NO: 1981 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1954;(ii) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1982, or that differs from SEQ ID NO: 1982 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1955; and(jj) a gRNA comprising a CRISPR RNA comprising a crRNA backbone comprising the nucleotide sequence set forth as SEQ ID NO: 1983, or that differs from SEQ ID NO: 1983 by 1 to 5 nucleotides, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1956.

174. The RGN system of any one of claims 165-173, wherein said target sequence is located adjacent to and 3' of a protospacer adjacent motif (PAM).

175. The RGN system of claim 174, wherein a) the RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO: 1 recognizes the PAM having a consensus nucleotide sequence set forth as AYG, wherein the PAM is adjacent to and 5' of the target sequence; b) the RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO: 2, 1933, or 1944, recognizes the PAM having a consensus nucleotide sequence set forth as ATTN, wherein the PAM is adjacent to and 5' of the target sequence; c) the RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO: 3, 1938, 1943, 1945, 1947, or 1949, recognizes the PAM having a consensus nucleotide sequence set forth as VTTN, wherein the PAM is adjacent to and 5' of the target sequence; d) the RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO: 4, 1930, 1932, 1934, 1936, 1939, 1940, 1941, 1942, 1946, 1948, 1952, 1954, 1955, or 1956, recognizes the PAM having a consensus nucleotide sequence set forth as TTN, wherein the PAM is adjacent to and 5' of the target sequence; e) the RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO: 1931 recognizes the PAM having a consensus nucleotide sequence set forth as STTN, wherein the PAM is adjacent to and 5' of the target sequence;f) the RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO: 1935 recognizes the PAM having a consensus nucleotide sequence set forth as TTH, wherein the PAM is adjacent to and 5' of the target sequence; g) the RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO: 1937 or 1950 recognizes the PAM having a consensus nucleotide sequence set forth as RTTN, wherein the PAM is adjacent to and 5' of the target sequence; h) the RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO: 1951 recognizes the PAM having a consensus nucleotide sequence set forth as RTYN, wherein the PAM is adjacent to and 5' of the target sequence; and i) the RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO: 1953 recognizes the PAM having a consensus nucleotide sequence set forth as ATG, wherein the PAM is adjacent to and 5' of the target sequence.

176. The RGN system of any one of claims 165-175, wherein the target sequence is within a cell.

177. The RGN system of claim 176, wherein the cell is a eukaryotic cell.

178. The RGN system of any one of claims 165-177, wherein the system directs cleavage of the target nucleic acid molecule.

179. The RGN system of claim 178, wherein the cleavage generates a double -stranded break.

180. The RGN system of claim 179, wherein the double -stranded break comprises:(i) a 5 nt to 12 nt 5’ overhang, wherein said RGN polypeptide has at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4; or(ii) a 1 nt 3 ’ overhang, wherein said RGN polypeptide has at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1.

181. The RGN system of any one of claims 165-177, wherein said RGN polypeptide is nuclease inactive.

182. The RGN system of claim 181, wherein said RGN polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to any one of SEQ ID NOs: 408, 597, 678, 827, 896, 979, 2686, and 2687.

183. The RGN system of any one of claims 165-182, wherein the RGN polypeptide is operably fused to a base -editing polypeptide or a polymerase editing polypeptide.

184. The RGN system of claim 183, wherein the base-editing polypeptide comprises a deaminase.

185. The RGN system of claim 183, wherein said polymerase editing polypeptide comprises a DNA polymerase or a reverse transcriptase.

186. The RGN system of any one of claims 165-185, wherein the RGN polypeptide comprises one or more nuclear localization signals (NLS).

187. The RGN system of any one of claims 165-186, wherein said system further comprises one or more donor polynucleotides.

188. A cell comprising the RGN system of any one of claims 165-187.

189. The cell of claim 192, wherein the cell is a eukaryotic cell.

190. A pharmaceutical composition comprising the nucleic acid molecule of any one of claims 1-57, and 161, the vector of any one of claims 58-87, and 162, the RGN polypeptide of any one of claims 94-146, the RNP complex of claim 147, the gRNA of any one of claims 148-160, the cell of any one of claims 88, 163, 164, 188, and 189, or the RGN system of any one of claims 165- 187, and a pharmaceutically acceptable carrier.

191. A method for binding a target sequence in a target nucleic acid molecule comprising delivering an RGN system according to any one of claims 165-187, to said target sequence or a cell comprising the target sequence.

192. A method for cleaving and / or modifying a target nucleic acid molecule comprising a target sequence, said method comprising delivering a system according to any one of claims 165-187 to said target sequence or a cell comprising the target sequence, wherein cleavage or modification of said target nucleic acid molecule occurs.

193. A method for binding one or more target sequence in a target nucleic acid molecule with an RNA-guided nuclease (RGN) polypeptide, said method comprising: a) assembling a ribonucleoprotein (RNP) complex by combining: i) a guide RNA (gRNA); and ii) an RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as any one of SEQ ID NOs: 1-4, 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 1087, 1105, 1108, 1116, 1121, 1122, 1123, 1126, 1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173, 1223, 1228, 1304, 1322, 1324, 1328, 1330, 1361, 1400, 1930-1956, 2686, and 2687; under conditions suitable for formation of the RNP complex; and b) contacting said target nucleic acid molecule or a cell comprising said target nucleic acid molecule with the assembled RNP complex; thereby binding said one or more target sequence with said RGN polypeptide.

194. A method for binding one or more target sequence in a target nucleic acid molecule with an RNA-guided nuclease (RGN) polypeptide, said method comprising contacting said targetnucleic acid molecule or a cell comprising said target nucleic acid molecule with i) a guide RNA (gRNA); and ii) an RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as any one of SEQ ID NOs: 1-4, 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 1087, 1105, 1108, 1116, 1121, 1122, 1123, 1126, 1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173, 1223, 1228, 1304, 1322, 1324, 1328, 1330, 1361, 1400, 1930-1956, 2686, and 2687, or a polynucleotide encoding said RGN polypeptide, thereby binding said target sequence with an RGN.

195. The method of claim 194, wherein the polynucleotide encoding said RGN polypeptide is an mRNA comprising a heterologous 5’ untranslated region (UTR) and / or a heterologous 3 ’ UTR.

196. The method of any one of claims 193-195, wherein said method is performed in vitro, in vivo, or ex vivo.

197. The method of any one of claims 193-196, wherein the cell is a eukaryotic cell.

198. The method of any one of claims 193-197, wherein said RGN polypeptide further comprises a base-editing polypeptide or a polymerase editing polypeptide, thereby allowing for modification of said target nucleic acid molecule.

199. The method of claim 198, wherein said base-editing polypeptide comprises a deaminase.

200. The method of claim 198, wherein said polymerase editing polypeptide comprises a DNA polymerase or a reverse transcriptase.

201. The method of any one of claims 193-197, wherein said RGN polypeptide is capable of cleaving said target nucleic acid molecule, thereby allowing for the cleaving and / or modifying of said target nucleic acid molecule.

202. The method of claim 201, wherein said cleaving generates a double -stranded break in said target nucleic acid molecule, wherein said double-stranded break comprises:(i) a 5 nt to 12 nt 5’ overhang, wherein said RGN polypeptide has at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4; or(ii) a 1 nt 3 ’ overhang, wherein said RGN polypeptide has at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1.

203. A method for cleaving and / or modifying a target nucleic acid molecule comprising one or more target sequence, said method comprising contacting the target nucleic acid molecule with:a) an RNA-guided nuclease (RGN) polypeptide, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as any one of SEQ ID NOs: 1-4, 408, 450, 463, 471, 481, 484, 485, 486, 500, 502, 505, 508, 539, 597, 602, 678, 707, 708, 717, 809, 810, 827, 866, 896, 898, 909, 912, 916, 960, 961, 963, 966, 970, 975, 979, 1012, 1019, 1034, 1051, 1056, 1059, 1064, 1069, 1070, 1072, 1074, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 1087, 1105, 1108, 1116, 1121, 1122, 1123, 1126, 1132, 1144, 1146, 1147, 1148, 1149, 1154, 1156, 1158, 1159, 1160, 1168, 1169, 1170, 1171, 1173, 1223, 1228, 1304, 1322, 1324, 1328, 1330, 1361, 1400, 1930-1956, 2686, and 2687; and b) a guide RNA (gRNA) capable of targeting the RGN polypeptide of (a) to a target sequence; wherein cleavage and / or modification of said target nucleic acid molecule occurs.

204. The method of claim 203, wherein cleavage by said RGN polypeptide generates a double -stranded break in said target DNA molecule, and wherein said double-stranded break comprises:(i) a 5 nt to 12 nt 5’ overhang, wherein said RGN polypeptide has at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4; or(ii) a 1 nt 3 ’ overhang, wherein said RGN polypeptide has at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1.

205. The method of claim 203, wherein said RGN polypeptide is nuclease inactive and is operably fused to a base -editing polypeptide or a polymerase editing polypeptide.

206. The method of claim 205, wherein the base-editing polypeptide comprises a deaminase.

207. The method of claim 205, wherein said polymerase editing polypeptide comprises a DNA polymerase or a reverse transcriptase.

208. The method of claim 203, wherein said modified target nucleic acid molecule comprises:(i) insertion of heterologous DNA into the target nucleic acid molecule;(ii) deletion of at least one nucleotide from the target nucleic acid molecule; or(iii) mutation of at least one nucleotide in the target nucleic acid molecule.

209. The method of any one of claims 203-208, wherein the target sequence is within a cell.

210. The method of claim 209, further comprising culturing the cell under conditions in which the RGN polypeptide is expressed and cleaves and / or modifies the target nucleic acid molecule to produce a modified target nucleic acid molecule; and selecting a cell comprising said modified target nucleic acid molecule.

211. A cell produced according to the method of claim 210, wherein said target nucleic acid molecule has been cleaved and / or modified at said target sequence.

212. The cell of claim 211, wherein the cell is a eukaryotic cell.

213. A pharmaceutical composition comprising the cell of claim 211 or 212 and a pharmaceutically acceptable carrier.