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, visualizing the target sequence of interest, and modifying the expression of the sequence of interest. The compositions include an RNA-guided nuclease polypeptide, a CRISPR RNA, a trans-activating CRISPR RNA, a guide RNA, and nucleic acid molecules encoding them. Vectors and host cells containing the nucleic acid molecules are also provided. Additionally, a CRISPR system for binding to a target sequence of interest is provided, the CRISPR system comprising an RNA-guided nuclease polypeptide and one or more guide RNAs. Methods and kits for detecting a target DNA sequence are also provided.

Description

[Technical field]

[0001] The present invention relates to the fields of molecular biology and gene editing.

[0002] Reference to sequence listing submitted as a text file via EFS-WEB This application contains a Sequence Listing submitted in ASCII format via EFS-Web, which is hereby incorporated by reference in its entirety. This ASCII copy, created on August 11, 2020, is assigned to L103438 1170WO 0049 2 Seq It is called List.txt and is 489,326 bytes in size. [Background technology]

[0003] Targeted genome editing or modification is becoming an important tool for basic and applied research. The first method was to engineer nucleases such as meganucleases, zinc finger fusion proteins or TALENs, which required the generation of chimeric nucleases with programmable sequence-specific 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 targeting of specific sequences by complexing the nuclease with a guide RNA that specifically hybridizes to a particular target sequence. Creating target-specific guide RNAs is less costly and more efficient than creating chimeric nucleases for each target sequence. Such RNA-guided nucleases can be used to optionally edit genomes through the introduction of sequence-specific double-strand breaks that are repaired via error-prone non-homologous end joining (NHEJ) and introduce mutations at specific genomic locations. Alternatively, heterologous DNA may be introduced into a genomic site via homology directed repair. Summary of the Invention

[0004] Compositions and methods are provided for binding a target sequence of interest. The compositions find use in cleaving or modifying a target sequence of interest, detecting a target sequence of interest, and modifying the expression of a sequence of interest. The compositions include an RNA-guided nuclease (RNAGN) polypeptide, a CRISPRRNA (crRNA), a trans-activating CRISPRRNA (tracrRNA), a guide RNA (gRNA), a nucleic acid molecule encoding the same, and a vector and a host cell comprising the nucleic acid molecule. A CRISPR system for binding a target sequence of interest is also provided, where the CRISPR system comprises an RNA-guided nuclease polypeptide and one or more guide RNAs. Thus, the methods disclosed herein are depicted for binding a target sequence of interest, and in some embodiments, for cleaving or modifying a target sequence of interest. The target sequence of interest can be modified, for example, as a result of non-homologous end joining or homologous directional repair with an introduced donor sequence. Additionally, methods and kits are provided for detecting a target DNA sequence of a DNA molecule using a detection single-stranded DNA. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0005] Many modifications and other embodiments of the inventions described 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. It is to be understood, therefore, that the invention is not 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.

[0006] I. Overview RNA-guided nucleases (RGNs) allow targeted manipulation of single sites within a genome and are useful in the context of gene targeting for therapeutic and research applications. In various organisms, including mammals, RNA-guided nucleases have been used for genome engineering, for example, by stimulating non-homologous end joining and homologous recombination. The compositions and methods described herein are useful for creating single- or double-stranded breaks in polynucleotides, modifying polynucleotides, detecting specific sites within polynucleotides, or modifying the expression of specific genes.

[0007] The RNA-guided nucleases disclosed herein can alter gene expression by modifying target sequences. In certain embodiments, the RNA-guided nucleases are directed to target sequences by guide RNAs (gRNAs) as part of a clustered regularly interspaced short palindromic repeats (CRISPR) RNA-guided nuclease system. The guide RNA forms a complex with the RNA-guided nuclease, binds the RNA-guided nuclease to the target sequence, and in some embodiments introduces a single-stranded or double-stranded break in the target sequence, so that RGNs are considered "RNA-guided." After the target sequence is cleaved, the break is repaired, and the DNA sequence of the target sequence is modified during the repair process. Thus, methods of using RNA-guided nucleases to modify target sequences in the DNA of a host cell are provided herein. For example, RNA-guided nucleases can be used to modify target sequences in genomic loci of eukaryotic or prokaryotic cells.

[0008] II. RNA-guided nucleases Provided herein is an RNA-guided nuclease. The term "RNA-guided nuclease" refers to a polypeptide that binds to a specific target nucleotide sequence in a sequence-specific manner and is directed to the target nucleotide sequence by a guide RNA molecule that is complexed with a polypeptide and hybridized with the target sequence. Although RNA-guided nucleases can cleave the target sequence upon binding, the term "RNA-guided nuclease" also includes nuclease-dead RNA-guided nucleases that bind but do not cleave the target sequence. The cleavage of the target sequence by RNA-guided nucleases can result in single-stranded or double-stranded cleavage. RNA-guided nucleases that can only cleave one strand of a double-stranded nucleic acid molecule are referred to herein as nickases.

[0009] RNA-guided nucleases disclosed herein include APG05733.1, APG06207.1, APG01647.1, APG08032.1, APG05712.1, APG01658.1, APG06498.1, APG09106.1, APG09882.1, APG02675.1, APG01405.1, APG06250.1, APG06877.1, APG09053.1, APG04293.1, APG01308.1, APG06646.1, APG09748, and APG07433.1. Included are RNA-guided nucleases whose amino acid sequences are set forth as SEQ ID NO: 1, 9, 16, 23, 30, 38, 46, 54, 61, 69, 75, 82, 89, 95, 103, 110, 117, 137 or 235, respectively, active fragments or variants thereof which retain the ability to bind to target nucleotide sequences in an RNA-guided, sequence-specific manner. In some of these embodiments, the active fragment or variant of APG05733.1, APG06207.1, APG01647.1, APG08032.1, APG05712.1, APG01658.1, APG06498.1, APG09106.1, APG09882.1, APG02675.1, APG01405.1, APG06250.1, APG06877.1, APG09053.1, APG04293.1, APG01308.1, APG06646.1, APG09748, or APG07433.1 RGN is capable of cleaving a single-stranded or double-stranded target sequence.In some embodiments, APG05733.1, APG06207.1, APG01647.1, APG08032.1, APG05712.1, APG01658.1, APG06498.1, APG09106.1, APG09882.1, APG02675.1, APG01405.1, APG06250.1, APG06877.1, APG09053.1, APG04293.1, APG01308.1, APG06646.1, APG09748, or APG07433.1 Active variants of RGN include amino acid sequences 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 the amino acid sequence set forth as SEQ ID NO:1, 9, 16, 23, 30, 38, 46, 54, 61, 69, 75, 82, 89, 95, 103, 110, 117, 137 or 235. In certain embodiments, APG05733.1, APG06207.1, APG01647.1, APG08032.1, APG05712.1, APG01658.1, APG06498.1, APG09106.1, APG09882.1, APG02675.1, APG01405.1, APG06250.1, APG06877.1, APG09053.1, APG04293.1, APG01308.1, APG06646.1, APG09748, or APG07433.1 An active fragment of RGN comprises at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050 or more consecutive amino acid residues of the amino acid sequence set forth in SEQ ID NO: 1, 9, 16, 23, 30, 38, 46, 54, 61, 69, 75, 82, 89, 95, 103, 110, 117, 137, or 235. The RNA-guided nucleases 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 the guide RNA.Additional domains that may be found in the RNA-guided nucleases provided herein include, but are not limited to, DNA-binding domains, helicase domains, protein-protein interaction domains, and dimerization domains. In certain embodiments, the RNA-guided nucleases provided herein may comprise at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% to one or more of a DNA-binding domain, a helicase domain, a protein-protein interaction domain, and a dimerization domain.

[0010] The target nucleotide sequence is bound by the RNA-guided nuclease provided herein and hybridizes with the guide RNA associated with the RNA-guided nuclease. The target sequence can then be cleaved by the RNA-guided nuclease if the polypeptide has nuclease activity. The term "cleavage" or "cleavage" refers to the hydrolysis of at least one phosphodiester bond in the backbone of the target nucleotide sequence, which can result in either a single-stranded or double-stranded break in the target sequence. The presently disclosed RGNs can cleave nucleotides in a polynucleotide functioning as an endonuclease, or can be an exonuclease, removing consecutive nucleotides from the ends (5' and / or 3' ends) of the polynucleotide. In other embodiments, the disclosed RGNs can cleave nucleotides of the target sequence in any position of the polynucleotide, thus functioning as both an endonuclease and an exonuclease. Cleavage of a target polynucleotide by the presently disclosed RGNs can result in a staggered cut or a blunt end.

[0011] The presently disclosed RNA-guided nuclease may be a wild-type sequence derived from a bacterial or archaeal species. Alternatively, the RNA-guided nuclease may be a mutant or fragment of a wild-type polypeptide. Wild-type RGN may be modified, for example, to change nuclease activity or change PAM specificity. In some embodiments, the RNA-guided nuclease is not naturally occurring.

[0012] In some embodiments, the RNA-guided nuclease functions as a nickase and only cleaves a single strand of the target nucleotide sequence. Such RNA-guided nucleases have a single functional nuclease domain. In some of these embodiments, the additional nuclease domain is mutated to reduce or eliminate nuclease activity.

[0013] In other embodiments, the RNA-guided nuclease completely lacks nuclease activity or exhibits reduced nuclease activity, referred to herein as nuclease-dead or nuclease-inactive. Any method known in the art for introducing mutations into amino acid sequences, such as PCR-mediated mutagenesis and site-directed mutagenesis, can be used to generate nickases or nuclease-dead RGNs. See, for example, U.S. Patent Application Publication No. 2014 / 0068797 and U.S. Patent No. 9,790,490, each of which is incorporated by reference in its entirety.

[0014] The RNA-guided nuclease lacking nuclease activity can be used to deliver fusion polypeptides, polynucleotides, or small molecule payloads to specific genomic locations. In some of these embodiments, the RGN polypeptide or guide RNA can be fused to a detectable label to allow detection of specific sequences. As a non-limiting example, the nuclease-dead RGN can be fused to a detectable label (e.g., a fluorescent protein) and targeted to specific sequences associated with disease to allow detection of disease-associated sequences.

[0015] Alternatively, nuclease-dead RGNs can be targeted to specific genomic locations to alter expression of desired sequences. In some embodiments, binding of the nuclease-dead RNA-guided nuclease to the target sequence results in suppression of expression of the target sequence or a gene under transcriptional control by the target sequence by preventing binding of RNA polymerase or transcription factors in the target genomic region. In other embodiments, the RGN (e.g., nuclease-dead RGN) or its complexed guide RNA further comprises an expression modulator that, upon binding to the target sequence, acts to either suppress or activate expression of the target sequence or a gene under transcriptional control by the target sequence. In some of these aspects, the expression modulator modulates expression of the target sequence or regulated gene via epigenetic mechanisms.

[0016] In other embodiments, nuclease-dead RGNs or RGNs with only nickase activity can target specific genomic locations to modify the sequence of a target polynucleotide via fusion to a base-editing polypeptide, such as a deaminase polypeptide or an active mutant or fragment thereof that deaminates a nucleotide base and results in the conversion of one nucleotide base to another. The base-editing polypeptide can be fused to the RGN at its N-terminus or C-terminus. Additionally, the base-editing polypeptide can be fused to the RGN via a peptide linker. Non-limiting examples of deaminase polypeptides useful in such compositions and methods include cytidine deaminase or adenosine deaminase (e.g., any of the adenosine deaminase base editing described in Gaudelli et al. (2017) Nature 551:464-471, US Patent Application Publication Nos. 2017 / 0121693 and 2018 / 0073012, and WO2018 / 027078, or the deaminases disclosed in PCT / US2019 / 068079, all of which are incorporated by reference herein in their entirety). Additionally, it is known in the art that certain fusion proteins between RGN and base editing enzymes may contain at least one uracil stabilizing polypeptide that increases the mutation rate of cytidine, deoxycytidine, or cytosine to thymidine, deoxythymidine, or thymine in a nucleic acid molecule by the deaminase. Non-limiting examples of uracil stabilizing polypeptides include those described in U.S. Provisional Application No. 63 / 052,175, filed July 15, 2020, and a uracil glycosylase inhibitor (UGI) domain (SEQ ID NO: 261) that can enhance base editing efficiency (U.S. Patent No. 10,167,547, incorporated herein by reference). Thus, a fusion protein may include at least one uracil stabilizing polypeptide, such as an RGN, or a variant thereof, a deaminase, and optionally a UGI, as described herein.

[0017] The RNA-guided nuclease fused to a polypeptide or domain may be separated or linked by a linker. The term "linker" as used herein refers to a chemical group or molecule that links two molecules or moieties, such as a binding domain and a cleavage domain of a nuclease. In some embodiments, the linker links the gRNA-binding domain of the RNA-guided nuclease and a base-editing polypeptide, such as a deaminase. In some embodiments, the linker links the nuclease-killing RGN and the deaminase. Typically, the linker is placed between or adjacent to two groups, molecules, or other moieties and is linked to each via a covalent bond, thus linking the two. In some embodiments, the linker is an amino acid or multiple amino acids (e.g., a peptide or protein). In some embodiments, the linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker is 1-5 amino acids in length, e.g., 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.

[0018] The currently disclosed RNA-guided nuclease can include at least one nuclear localization signal (NLS) to enhance the transport of RGN to the nucleus of a cell. Nuclear localization signals are known in the art and generally include a series of basic amino acids (e.g., Lange et al., J. Biol. Chem. (2007) 282:5101-5105). In certain embodiments, RGN includes two, three, four, five, six or more nuclear localization signals. The nuclear localization signal can be a heterologous NLS. Non-limiting examples of nuclear localization signals useful for the currently disclosed RGN are the nuclear localization signals of SV40 large T antigen, nucleopasmin, and c-Myc (see, e.g., Ray et al. (2015) Bioconjug Chem 26(6):1004-7). In certain embodiments, RGN includes the NLS sequence shown in SEQ ID NO: 125 or 127. RGN can contain one or more NLS sequences at its N-terminus, C-terminus, or both N-terminus and C-terminus, for example, RGN can contain two NLS sequences in the N-terminal region and four NLS sequences in the C-terminal region.

[0019] Other localization signal sequences known in the art that localize a polypeptide to a particular subcellular location can also be used to target RGN, including, but not limited to, plastid localization sequences, mitochondrial localization sequences, and dual targeting signal sequences that target both plastids and mitochondria (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; Soll (2002) Curr Opin Plant Biol 5:529-535; Carrie and Small (2013) Biochim Biophys Acta 1833:253-259; Carrie et al. (2009) (See 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).

[0020] In certain embodiments, the presently disclosed RNA-guided nuclease comprises at least one cell-penetrating domain that facilitates cellular uptake of RGN. Cell-penetrating domains are known in the art and generally comprise a stretch of positively charged amino acid residues (i.e., polycationic cell-penetrating domains), alternating polar and non-polar amino acid residues (i.e., amphipathic cell-penetrating domains), or hydrophobic amino acid residues (i.e., hydrophobic cell-penetrating domains) (see, for example, Milletti F. (2012) Drug Discov Today 17:850-860). A non-limiting example of a cell-penetrating domain is the transactivating transcription activator (TAT) from human immunodeficiency virus 1.

[0021] The nuclear localization signal, plastid localization signal, mitochondrial localization signal, dual targeting localization signal, and / or cell penetrating domain can be located at the amino terminus (N-terminus), carboxyl terminus (C-terminus), or at an internal position of the RNA-guided nuclease.

[0022] The presently disclosed RGN can be fused directly or indirectly via a linker peptide to an effector domain, such as a cleavage domain, a deaminase domain, or an expression modulator domain. Such domains can be located at the N-terminus, C-terminus, or internal position of the RNA-guided nuclease. In some of these embodiments, the RGN component of the fusion protein is a nuclease-dead RGN.

[0023] In some embodiments, the RGN fusion protein is any domain capable of cleaving polynucleotides (i.e., RNA, DNA, or RNA / DNA hybrids), including cleavage domains including, but not limited to, restriction endonucleases and homing nucleases, such as type IIS endonucleases (e.g., FokI) (Belfort et al. (1997) Nucleic Acids Res. 25:3379-3388; Linn et al. (eds.) Nucleases, Cold Spring Harbor Laboratory Press, 1993).

[0024] In other embodiments, the RGN fusion protein comprises a deaminase domain that deaminates a nucleotide base, resulting in the conversion of one nucleotide base to another, including, but not limited to, cytidine deaminase or adenosine deaminase base editing (e.g., Gaudelli et al. (2017) Nature 551:464-471; U.S. Patent Application Publication Nos. 2017 / 0121693 and 2018 / 0073012, U.S. Patent No. 9,840,699, and International Publication No. WO2018 / 027078).

[0025] In some embodiments, the effector domain of the RGN fusion protein can be an expression modulator domain, which is a domain that serves to upregulate or downregulate transcription. The expression modulator domain can be an epigenetic modification domain, a transcriptional repressor domain, or a transcriptional activation domain.

[0026] In some of these embodiments, the expression modulator of the RGN fusion protein comprises an epigenetic modification domain that covalently modifies DNA or histone proteins to change histone and / or chromosomal structure without changing the DNA sequence, resulting in altered gene expression (i.e., up-regulation or down-regulation). 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, as well as methylation and hydroxymethylation of cytosine residues in DNA. Non-limiting examples of epigenetic modification domains include histone acetyltransferase domains, histone deacetylase domains, histone methyltransferase domains, histone demethylase domains, DNA methyltransferase domains, and DNA demethylase domains.

[0027] In other embodiments, the expression modulator of the fusion protein comprises a transcriptional repressor domain that interacts with transcriptional control elements and / or transcriptional regulatory proteins, such as RNA polymerase 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, Sp1-like repressors, IκB, and KrUppel-associated box (KRAB) domains.

[0028] In yet other embodiments, the expression modulator of the fusion protein comprises a transcriptional activation domain that interacts with transcriptional control elements and / or transcriptional regulatory proteins, such as RNA polymerase 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, herpes simplex virus VP16 activation domain and NFAT activation domain.

[0029] The presently disclosed RGN polypeptide can include a detectable label or purification tag. The detectable label or purification tag can be located at the N-terminus, C-terminus, or internal position of the RNA-guided nuclease, directly or indirectly via a linker peptide. In some of these embodiments, the RGN component of the fusion protein is a nuclease-dead RGN. In other embodiments, the RGN component of the fusion protein is an RGN with nickase activity.

[0030] Detectable labels are molecules that can be visualized or otherwise observed. Detectable labels can be fused to RGN as a fusion protein (e.g., fluorescent protein) or can be small molecules conjugated to RGN polypeptides that can be detected visually or by other means. Detectable labels that can be fused to RGN presently disclosed as fusion proteins include, but are not limited to, fluorescent proteins or any detectable protein domain, including a protein domain that can be detected with a specific antibody. Non-limiting examples of fluorescent proteins include green fluorescent protein (e.g., GFP, EGFP, ZsGreen1) and yellow fluorescent protein (e.g., YFP, EYFP, ZsYellow1). Non-limiting examples of small molecule detectable labels include: 3 H and 35 Examples of such radiolabels include S.

[0031] RGN polypeptides can also include a purification tag, which is any molecule that can be used to isolate a protein or fusion protein from a mixture (e.g., biological sample, culture medium). Non-limiting examples of purification tags include biotin, myc, maltose binding protein, and glutathione-S-transferase.

[0032] II. Guide RNA The present disclosure provides guide RNAs and polynucleotides encoding the same. The term "guide RNA" refers to a nucleotide sequence that hybridizes with a target sequence and has sufficient complementarity with a target nucleotide sequence for direct sequence-specific binding of an associated RNA-guided nuclease to the target nucleotide sequence. Thus, each guide RNA of an RGN is one or more RNA molecules (generally one or two) that can bind to the RGN and induce the RGN to bind to a specific target nucleotide sequence, and if the RGN has a nickase or nuclease activity, can also cleave the target nucleotide sequence. Generally, guide RNAs include CRISPRRNAs (crRNAs), and in some embodiments, trans-activating CRISPRRNAs (tracrRNAs). Natural guide RNAs, including both crRNAs and tracrRNAs, generally include two separate RNA molecules that hybridize to each other via the repeat sequence of the crRNA and the anti-repeat sequence of the tracrRNA.

[0033] The naturally occurring direct repeat sequences in a CRISPR array generally range from 28 to 37 base pairs in length, but the length can vary between about 23 bp and about 55 bp. The spacer sequences in a CRISPR array generally range from about 32 to about 38 bp in length, but the length can be between about 21 bp and about 72 bp. Each CRISPR array generally contains less than 50 units of CRISPR repeat spacer sequences. CRISPRs are transcribed as part of a long transcript called the primary CRISPR transcript, which constitutes the majority of the CRISPR array. The primary CRISPR transcript is cleaved by Cas proteins to generate crRNA or, in some cases, pre-crRNA. The pre-crRNA is further processed into mature crRNA by additional Cas proteins. The mature crRNA contains the spacer sequence and the CRISPR repeat sequence. In some embodiments, where the pre-crRNA is processed into mature (or processed) crRNA, maturation involves removal of about 1 to about 6 or more 5', 3', or 5' and 3' nucleotides. These nucleotides removed during maturation of the pre-crRNA molecule are not necessary for generating or designing a guide RNA for the purposes of genome editing or targeting a specific target nucleotide sequence of interest.

[0034] CRISPR RNA (crRNA) comprises a spacer sequence and a CRISPR repeat sequence. A "spacer sequence" is a nucleotide sequence that directly hybridizes to a target nucleotide sequence of interest. The spacer sequence is engineered to be fully or partially complementary to the target sequence of interest. In various embodiments, the spacer sequence can comprise from about 8 nucleotides to about 30 nucleotides, or more. For example, the spacer sequence 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 degree of complementarity between a spacer sequence and its corresponding target sequence is 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, optionally when aligned using a suitable alignment algorithm. In certain embodiments, the spacer sequence does not contain 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(1):23-24).

[0035] The CRISPR RNA repeat sequence comprises a nucleotide sequence that comprises a region having sufficient complementarity to hybridize to tracrRNA. In various embodiments, the CRISPR RNA repeat sequence can comprise about 8 nucleotides to about 30 nucleotides, or more. For example, the CRISPR repeat sequence 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 in length. In one embodiment, the CRISPR repeat sequence is about 21 nucleotides in length. In some embodiments, the degree of complementarity between a CRISPR repeat sequence and its corresponding tracrRNA sequence is 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, optionally when aligned using a suitable alignment algorithm. In certain embodiments, the CRISPR repeat sequence comprises the nucleotide sequence of SEQ ID NO: 2, 10, 17, 24, 31, 39, 47, 55, 62, 70, 76, 83, 90, 96, 104, 111, 118, 240, 273, or 287, or an active variant or fragment thereof that, when contained within a guide RNA, is capable of directing sequence-specific binding of an associated RNA-guided nuclease provided herein to a target sequence of interest. In certain embodiments, an active CRISPR repeat 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 identity to the nucleotide sequence set forth as SEQ ID NO: 2, 10, 17, 24, 31, 39, 47, 55, 62, 70, 76, 83, 90, 96, 104, 111, 118, 240, 273 or 287.In certain embodiments, an active CRISPR repeat fragment of a wild type sequence comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or more contiguous nucleotides of the nucleotide sequence set forth as SEQ ID NO: 2, 10, 17, 24, 31, 39, 47, 55, 62, 70, 76, 83, 90, 96, 104, 111, 118, 240, 273, or 287.

[0036] In some embodiments, the crRNA is not naturally occurring. In some of these embodiments, the specific CRISPR repeat sequence is not essentially linked to the engineered spacer sequence, and the CRISPR repeat sequence is considered to be heterologous to the spacer sequence. In some embodiments, the spacer sequence is a non-naturally occurring engineered sequence.

[0037] A transactivating CRISPR RNA or tracrRNA molecule comprises a nucleotide sequence that comprises a region that has sufficient complementarity to hybridize with the CRISPR repeat sequence of the crRNA, which is referred to herein as the anti-repeat region. In some embodiments, the tracrRNA molecule further comprises a region that has a secondary structure (e.g., a stem-loop) or forms a secondary structure by hybridizing with the corresponding crRNA. In certain embodiments, the region of the tracrRNA that is fully or partially complementary to the CRISPR repeat sequence is the 5' end of the molecule, and the 3' end of the tracrRNA contains a secondary structure. This secondary structure region generally contains several hairpin structures, including a nexus hairpin found adjacent to the anti-repeat sequence. The nexus hairpin often has a conserved nucleotide sequence at the base of the hairpin stem with the motifs UNANNA, CNANNG, CNANNU, UNANNG, UNANNC, or CNANNU (SEQ ID NOs: 8, 37, 45, 53, 68, and 102, respectively), which are found in many nexus hairpins of tracrRNA. The 3' end of tracrRNA often has terminal hairpin structures that vary in structure and number, but often contain a GC-rich Rho-independent transcription terminator hairpin followed by a string of 3' terminal U's. See, e.g., Briner et al. (2014) Molecular Cell 56:333-339, Briner and Barrangou (2016) Cold Spring Harb Protoc; doi: 10.1101 / pdb.top090902, and US Publication No. 2017 / 0275648, each of which is incorporated by reference in its entirety.

[0038] In various embodiments, the anti-repeat region of the tracrRNA that is fully or partially complementary to the CRISPR repeat sequence comprises from about 8 nucleotides to about 30 nucleotides, or more. For example, the region of base pairing between the tracrRNA anti-repeat sequence and the CRISPR repeat sequence 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 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, or more nucleotides in length. In certain embodiments, the anti-repeat region of the tracrRNA that is fully or partially complementary to the CRISPR repeat sequence is about 20 nucleotides in length. In some embodiments, the degree of complementarity between a CRISPR repeat sequence and its corresponding tracrRNA sequence is 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, optionally when aligned using a suitable alignment algorithm.

[0039] In various embodiments, the entire tracrRNA can comprise about 60 nucleotides to about 140 nucleotides. For example, the tracrRNA can be about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, or more nucleotides in length. In certain embodiments, the tracrRNA is about 80 to about 90 nucleotides in length, including about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, and about 90 nucleotides in length. In some embodiments, the tracrRNA is about 85 nucleotides in length.

[0040] In certain embodiments, the tracrRNA comprises the nucleotide sequence of SEQ ID NO: 3, 11, 18, 25, 32, 40, 48, 56, 63, 71, 77, 84, 91, 97, 105, 112, or 119, or an active variant or fragment thereof that, when contained within a guide RNA, can direct sequence-specific binding of an associated RNA-guided nuclease provided herein to a target sequence of interest. In certain embodiments, an active tracrRNA sequence 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 the nucleotide sequence set forth as SEQ ID NO: 3, 11, 18, 25, 32, 40, 48, 56, 63, 71, 77, 84, 91, 97, 105, 112, 119, 241, 274, or 286. In certain embodiments, an active tracrRNA sequence fragment of a wild type sequence comprises at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 or more contiguous nucleotides relative to the nucleotide sequence set forth as SEQ ID NO: 3, 11, 18, 25, 32, 40, 48, 56, 63, 71, 77, 84, 91, 97, 105, 112, 119, 241, 274, or 286.

[0041] Two polynucleotide sequences can be considered substantially complementary if the two sequences hybridize to each other under stringent conditions. Similarly, if a guide RNA bound to an RGN binds to a target sequence under stringent conditions, the RGN is considered to bind to a particular target sequence in a sequence-specific manner. "Stringent conditions" or "stringent hybridization conditions" refer to conditions under which two polynucleotide sequences hybridize to each other to a detectably higher degree than other sequences (e.g., at least twice background). Stringent conditions are sequence-dependent and will be different in different circumstances. Typically, stringent conditions are at pH 7.0-8.3, with a salt concentration of about 1.5 M Na ion, typically less than about 0.01-1.0 M Na ion concentration (or other salt), and a temperature of at least about 30° C. for short sequences (e.g., 10-50 nucleotides); for long sequences (e.g., more than 50 nucleotides), stringent conditions can also be achieved by adding destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer of 30-35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate) at 37° C., and washing with 1×-2×SSC (20×SSC=3.0 M NaCl / 0.3 M trisodium citrate) at 50-55° C. Exemplary medium stringency conditions include hybridization with 40-45% formamide, 1.0 M NaCl, 1% SDS at 37° C., and washing with 0.5×-1×SSC at 55-60° C. Exemplary high stringency conditions include hybridization with 50% formamide, 1 M NaCl, 1% SDS at 37° C., and washing with 0.1×SSC at 60-65° C. Optionally, the wash buffer may contain about 0.1% to about 1% SDS. The duration of hybridization is generally less than about 24 hours, and usually from about 4 to about 12 hours. The duration of the wash period is at least long enough to reach equilibrium.

[0042] 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, Tm can be approximated from the formula of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284: Tm=81.5°C+16.6(log M)+0.41(%GC)-0.61(%form)-0.61(%form)-500 / L, where M is the molar concentration of monovalent cations, %GC is the percentage of guanosine and cytosine nucleotides in DNA, %form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. In general, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for a particular sequence and its complementarity at a defined ionic strength and pH. However, highly stringent conditions may utilize hybridization and / or washing at 1, 2, 3, or 4° C. below the thermal melting point (Tm). Moderately stringent conditions may utilize hybridization and / or washing at 6, 7, 8, 9, or 10° C. below the thermal melting point (Tm). Low stringency conditions may utilize hybridization and / or washing at 11, 12, 13, 14, 15, or 20° C. below the thermal melting point (Tm). Using this formula, hybridization and washing compositions, and the desired Tm, one of skill in the art will understand that variations in stringency of hybridization and / or washing solutions are essentially described.Extensive guides to nucleic acid hybridization can be 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).

[0043] The guide RNA may be a single guide RNA or a dual guide RNA system. A single guide RNA comprises a crRNA and a tracrRNA on a single RNA molecule, whereas a dual guide RNA system comprises a crRNA and a tracrRNA present on two different RNA molecules and hybridized to each other via at least a portion of the CRISPR repeat sequence of the crRNA and at least a portion of the tracrRNA that may be fully or partially complementary to the CRISPR repeat sequence of the crRNA. In some embodiments where the guide RNA is a single guide RNA, the crRNA and the tracrRNA are separated by a linker nucleotide sequence. Generally, the linker nucleotide sequence is one that does not contain complementary bases to avoid the formation of secondary structures within or including the nucleotides of the linker nucleotide sequence. In some embodiments, the linker nucleotide sequence between the crRNA and the tracrRNA is at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12 nucleotides in length, or more. In certain embodiments, the linker nucleotide sequence of the single guide RNA is at least 4 nucleotides in length. In one embodiment, the linker nucleotide sequence is the nucleotide sequence shown as SEQ ID NO:123.

[0044] Single or dual guide RNAs can be synthesized chemically or via in vitro transcription. Assays for determining sequence-specific binding between RGN and guide RNA are known in the art, including, but not limited to, in vitro binding assays between expressed RGN and guide RNA, which can be tagged with a detectable label (e.g., biotin) and used in pull-down detection assays to capture guide RNA:RGN complexes via detectable labels (e.g., streptavidin beads). A control guide RNA with a sequence or structure unrelated to the guide RNA can be used as a negative control for non-specific binding of RGN to RNA. In certain embodiments, the guide RNA is SEQ ID NO: 4, 12, 19, 26, 33, 41, 49, 57, 64, 72, 78, 85, 92, 98, 106, 113, or 120, and the spacer is a sequence of any sequence, indicated as a poly-N sequence.

[0045] In some aspects, the guide RNA can be introduced into a target cell, organelle, or embryo as an RNA molecule. The guide RNA can be in vitro transcribed or chemically synthesized. In other embodiments, a nucleotide sequence encoding the guide RNA is introduced into a cell, organelle, or embryo. In some of these aspects, the nucleotide sequence encoding the guide RNA is operably linked to a promoter (e.g., an RNA polymerase III promoter). The promoter may be a native promoter or may be heterologous to the nucleotide sequence encoding the guide RNA.

[0046] In various embodiments, the guide RNA can be introduced into a target cell, organelle, or embryo as a ribonucleoprotein complex, as described herein, where the guide RNA is bound to an RNA-guided nuclease polypeptide.

[0047] The guide RNA directs the associated RNA-guided nuclease to a specific target nucleotide sequence of interest via hybridization of the guide RNA to the target nucleotide sequence. The target nucleotide sequence may comprise DNA, RNA, or a combination of both, and may be single-stranded or double-stranded. The target nucleotide sequence may be genomic DNA (i.e., chromosomal DNA), plasmid DNA, or an RNA molecule (e.g., messenger RNA, ribosomal RNA, transfer RNA, microRNA, small interfering RNA). The target nucleotide sequence may be bound (and in some embodiments cleaved) by the RNA-guided nuclease in vitro or in a cell. The chromosomal sequence targeted by the RGN may be a nuclear, plastid, or mitochondrial chromosomal sequence. In some embodiments, the target nucleotide sequence is unique in the target genome.

[0048] The target nucleotide sequence is adjacent to a protospacer adjacent motif (PAM). The protospacer adjacent motif is generally within about 1 to about 10 nucleotides of the target nucleotide sequence, including about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 nucleotides from the target nucleotide sequence. The PAM can be 5' or 3' of the target sequence. In some embodiments, the PAM is 3' of the target sequence of the presently disclosed RGN. Generally, the PAM is a consensus sequence of about 3-4 nucleotides, but in certain embodiments, it can be 2, 3, 4, 5, 6, 7, 8, 9, or more nucleotides in length. In various embodiments, the PAM sequences recognized by the presently disclosed RGNs include the consensus sequences shown as SEQ ID NOs: 7, 15, 22, 29, 36, 44, 52, 60, 67, 81, 88, 101, 109, or 116.

[0049] In certain embodiments, an RNA-guided nuclease having SEQ ID NO: 1, 9, 16, 23, 30, 38, 46, 54, 61, 69, 75, 82, 89, 95, 103, 110, or 117, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to the PAM sequence set forth in SEQ ID NO: 7, 15, 22, 29, 36, 44, 52, 60, 67, 81, 88, 101, 109, or 116, respectively. In some embodiments, an RNA-guided nuclease having SEQ ID NO: 54 or 137, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to the PAM sequence set forth as SEQ ID NO: 147. In some of these embodiments, the RGN binds to a guide sequence comprising a CRISPR repeat sequence as set forth in SEQ ID NO: 2, 10, 17, 24, 31, 39, 47, 55, 62, 70, 76, 83, 90, 96, 104, 111, or 118, or an active variant or fragment thereof, and a tracrRNA sequence as set forth in SEQ ID NO: 3, 11, 18, 25, 32, 40, 48, 56, 63, 71, 77, 84, 91, 97, 105, 112, or 119, or an active variant or fragment thereof. The RGN system is further described in Example 1 and Tables 1 and 2 herein.

[0050] 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). This can be altered by modifying the promoter used to express the RGN or the amount of ribonucleoprotein complex delivered to a cell, organelle, or embryo.

[0051] When RGN recognizes its corresponding PAM sequence, it can cleave the target nucleotide sequence at a specific cleavage site. As used herein, a cleavage site is composed of two specific nucleotides in the target nucleotide sequence where the nucleotide sequence is cleaved by RGN. The cleavage site can include the first and second, second and third, third and fourth, fourth and fifth, fifth and sixth, seventh and eighth, or eighth and ninth nucleotides from the PAM in either the 5' or 3' direction. In some embodiments, the cleavage site can be more than 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the PAM in either the 5' or 3' direction. In some embodiments, the cleavage site is 4 nucleotides away from the PAM. In other embodiments, the cleavage site is at least 15 nucleotides away from the PAM. Because RGNs can cleave a target nucleotide sequence, generating a staggered end, in some embodiments, the cleavage site is defined based on a distance of two nucleotides from the PAM on the positive (+) strand of the polynucleotide and a distance of two nucleotides from the PAM on the negative (-) strand of the polynucleotide.

[0052] PAM is considered a hallmark of RNA-guided nucleases in type II CRISPR systems (Szczelkun et al., PNAS, 111: 9798-9803, 2014; Sternberg et al., Nature 507: 62-67, 2014). Interestingly, although APG06646.1 and APG04293.1 function as RNA-guided nucleases and have many of the same domains as type II CRISPR Cas9 nucleases, they each lack the typical PAM-interacting domain (PID; Interpro: IPR032237; Pfam: PF16595). Thus, APG06646.1 and APG04293.1 also do not have the typical PAM requirement, which is a 2-5 nucleotide motif as described above. Instead, these proteins possess unique DNA recognition domains at their C-termini (residues 821-1092 of APG06646.1 (full length sequence SEQ ID NO:117); residues 1064-1401 of APG04293.1 (full length sequence SEQ ID NO:103). These unique DNA recognition domains enable the nucleases to cleave at genomic target sites based on single nucleotide motifs in the vicinity of the genomic target sequence (SEQ ID NO:109; see Table 2).

[0053] APG04293.1 also possesses a unique signature domain of 133 amino acid residues proximal to its N-terminus (residues 144-276), the function of which is unknown in Type II CRISPR Cas9 nucleases or in the art in general.

[0054] III. Nucleotides encoding RNA-guided nucleases, CRISPR RNAs and / or tracrRNAs The present disclosure provides polynucleotides comprising the presently disclosed CRISPR RNA, tracrRNA, and / or sgRNA, as well as polynucleotides comprising nucleotide sequences encoding the presently disclosed RNA-guided nucleases, CRISPR RNA, tracrRNA, and / or sgRNA. The presently disclosed polynucleotides include those that comprise or encode CRISPR repeat sequences comprising the nucleotide sequences of SEQ ID NOs: 2, 10, 17, 24, 31, 39, 47, 55, 62, 70, 76, 83, 90, 96, 104, 111, 118, 240, 273, or 287, or active variants or fragments thereof that, when contained within the guide RNA, can direct sequence-specific binding of an associated RNA-guided nuclease to a target sequence of interest. Also disclosed are polynucleotides comprising or encoding tracrRNAs comprising the nucleotide sequence of SEQ ID NO: 3, 11, 18, 25, 32, 40, 48, 56, 63, 71, 77, 84, 91, 97, 105, 112, 119, 241, 274, or 286, or active variants or fragments thereof that, when contained within a guide RNA, can direct sequence-specific binding of an associated RNA-guided nuclease to a target sequence of interest. Also provided are polynucleotides encoding RNA-guided nucleases comprising the amino acid sequence set forth as SEQ ID NO: 1, 9, 16, 23, 30, 38, 46, 54, 61, 69, 75, 82, 89, 95, 103, 110, 117, 137, or 235, and active fragments or variants thereof that retain the ability to bind to a target nucleotide sequence in an RNA-guided sequence-specific manner.

[0055] The use of the term "polynucleotide" is not intended to limit the present disclosure to polynucleotides that contain DNA. Those skilled in the art will recognize that polynucleotides can include ribonucleotides, and combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogs. These include peptide nucleic acids (PNAs), PNA-DNA chimers, locked nucleic acids (LNAs), and phosphothiolate-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, and the like.

[0056] 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 whose codon usage frequency is designed to mimic the frequency of preferred codon usage or transcription conditions of a particular host cell. Expression in a particular host cell or organism is enhanced as a result of one or more codon changes at the nucleic acid level such that the translated amino acid sequence is unchanged. Nucleic acid molecules can be fully or partially codon optimized. Codon tables and other references providing preference information for a wide range of organisms are available in the art (see, for example, Campbell and Gowri (1990) Plant Physiol. 92:1-11 for plant preferred codon usage). Methods are available in the art for synthesizing plant-preferred genes. See, e.g., U.S. Pat. Nos. 5,380,831, 5,436,391, and Murray et al. (1989) Nucleic Acids Res. 17:477-498, which are incorporated herein by reference.

[0057] Polynucleotides encoding the RNA, crRNA, tracrRNA, and / or sgRNA provided herein may be provided in an expression cassette for expression in vitro or in a cell, organelle, embryo, or organism of interest. The cassette will include 5' and 3' regulatory sequences operably linked to the polynucleotides encoding the RGN, crRNA, tracrRNA, and / or sgRNA provided herein that allow for expression of the polynucleotide. The cassette may further include at least one additional gene or genetic element that is cotransformed into the organism. When an additional gene or element is 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., a region encoding an RGN, crRNA, tracrRNA, and / or sgRNA) is a functional linkage 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 linkage of two protein coding regions, operably linked means that the coding regions are in the same reading frame. Alternatively, additional genes or elements can be provided on multiple expression cassettes. For example, the nucleotide sequence encoding the presently disclosed RGN can be present on one expression cassette, while the nucleotide sequence encoding the crRNA, tracrRNA, or complete guide RNA can be present on a separate expression cassette. Such expression cassettes are provided with multiple restriction and / or recombination sites for insertion of polynucleotides under the transcriptional control of the regulatory regions. The expression cassette can further comprise a selectable marker gene.

[0058] The expression cassette comprises, in the 5'-3' direction of transcription, a transcriptional (and, in some embodiments, translational) initiation region (i.e., promoter) of the invention, an RGN-, crRNA-, tracrRNA- and / or sgRNA-encoding polynucleotide, and a transcriptional (and, in some embodiments, translational) termination region that is functional in the organism of interest. The promoter of the invention can direct or drive expression of a coding sequence in a host cell. The regulatory regions (e.g., promoter, transcriptional regulatory region, and translational termination region) may be endogenous or heterologous to the host cell or to each other. As used herein, "heterologous" with respect to a sequence is a sequence that originates from a foreign species or, if originating from the same species, is substantially altered 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 transcriptional initiation region that is heterologous to the coding sequence.

[0059] Convenient termination regions are available from the Ti plasmid of A. tumefaciens, e.g., 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 al. (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.

[0060] Additional regulatory signals include, but are not limited to, transcription initiation sites, operators, activators, enhancers, other regulatory elements, ribosome binding sites, start codons, termination signals, etc. See, e.g., U.S. Patent Nos. 5,039,523 and 4,853,331; European Patent No. 0480762A2; Sambrook et al. (1992) Molecular Cloning: A Laboratory Manual, ed. Maniatis et al. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) (hereinafter "Sambrook 11"); Davis et al., eds. (1980) Advanced Bacterial Genetics (Cold Spring Harbor Laboratory Press), Cold Spring Harbor, NY, and references cited therein.

[0061] In preparing an expression cassette, various DNA fragments can be manipulated to provide DNA sequences in the proper orientation and, where appropriate, in the proper reading frame. To this end, adapters or linkers can be used to join DNA fragments or other manipulations to provide convenient restriction sites, removal of unwanted DNA, removal of restriction sites, etc. To this end, in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions, can be involved.

[0062] Numerous promoters can be used in the practice of the present invention. The promoter can be selected based on the desired outcome. The nucleic acid can be combined with constitutive, inducible, developmental stage specific, cell type specific, tissue preferred, tissue specific, or other promoters for expression in the organism of interest. See, for example, WO 99 / 43838, and the promoters described in U.S. Patent Nos. 8,575,425; 7,790,846; 8,147,856; 8,586,832; 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, which are incorporated herein by reference.

[0063] For expression in plants, constitutive promoters also include the 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 al. (1992) Plant Mol. Biol. 18:675-689); pEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588); and MAS (Velten et al. (1984) EMBO J. 3:2723-2730).

[0064] Examples of inducible promoters are the Adh1 promoter induced by hypoxia or cold stress, the Hsp70 promoter induced by heat stress, the PPDK promoter, and the peptcarboxylase promoter induced by light. Also useful are chemically inducible promoters, such as the safener-inducible In2-2 promoter (U.S. Pat. No. 5,364,780), the Axig1 promoter (auxin-inducible and tapetum-specific but also active in callus) (PCT / US01 / 22169), steroid-responsive promoters (e.g., the estrogen-inducible ERE 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), and tetracycline-inducible and tetracycline-repressible promoters (see, e.g., Gatz et al. (1991) Mol. Gen. Genet. 227:229-237; U.S. Pat. Nos. 5,814,618 and 5,789,156), which are incorporated herein by reference.

[0065] Tissue-specific or tissue-preferred promoters can be used to target expression of an expression construct in a particular tissue. In some embodiments, tissue-specific or tissue-preferred promoters are active in plant tissues. Examples of promoters under plant developmental control include promoters that preferentially initiate transcription in specific tissues, such as leaves, roots, fruits, seeds, and flowers. A "tissue-specific" promoter is a promoter that initiates transcription only in a particular tissue. Unlike constitutive expression of a gene, tissue-specific expression is the result of the interaction of several levels of gene regulation. Thus, promoters from homologous or closely related plant species are preferably used to achieve efficient and reliable expression of transgenes in a particular tissue. In some embodiments, the expression comprises a tissue-preferred promoter. A "preferred tissue" promoter is a promoter that selectively initiates transcription in a particular tissue, but not necessarily entirely or exclusively.

[0066] In some embodiments, the nucleic acid molecules encoding RGN, crRNA, and / or tracrRNA comprise a cell type specific promoter. A "cell type specific" promoter is a promoter that drives expression primarily in a particular cell type in one or more organs. Some examples of plant cells in which a cell type specific promoter functional in plants may be primarily active include, for example, BETL cells, root vascular cells, leaves, stem cells, and stem cells. The nucleic acid molecule may also comprise a cell type preferred promoter. A "cell type preferred" promoter is a promoter that drives expression primarily, but not necessarily entirely or exclusively, in a particular cell type in one or more organs. Some examples of plant cells in which a cell type preferred promoter in plants may be selectively active include, for example, BETL cells, root vascular cells, leaves, stem cells, and stem cells.

[0067] The nucleic acid sequences encoding the RNA, crRNA, tracrRNA, and / or sgRNA can be operably linked to a promoter sequence recognized by a phage RNA polymerase, such as in vitro mRNA synthesis. In such embodiments, the in vitro transcribed 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 variant of a T7, T3, or SP6 promoter sequence. In such embodiments, the expressed protein and / or RNA can be purified for use in the methods of genome modification described herein.

[0068] In certain embodiments, the polynucleotides encoding RGN, crRNA, tracrRNA, and / or sgRNA may also be linked to a polyadenylation signal (e.g., SV40 polyA signal and other functional signals in plants) and / or at least one transcription termination sequence. Furthermore, the sequence encoding RGN may also be linked to a sequence encoding at least one nuclear localization signal, at least one cell penetration domain, and / or at least one signal peptide capable of transporting the protein to a specific intracellular location, as described elsewhere herein.

[0069] The polynucleotides encoding RGN, crRNA, tracrRNA, and / or sgRNA can be present in a vector or vectors. "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 / minichromosomes, transposons, and viral vectors (e.g., lentiviral vectors, adeno-associated viral vectors, baculoviral vectors). Vectors can include additional expression control sequences (e.g., enhancer sequences, Kozak sequences, polyadenylation sequences, transcription 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, NY, 3rd edition, 2001.

[0070] The vector may also contain a selectable marker gene for the selection of transformed cells. The selectable marker gene is utilized for the selection of transformed cells or tissues. Marker genes include genes encoding antibiotic resistance, such as neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), and genes conferring resistance to herbicidal compounds, such as glufosinate ammonium, bromoxynil, imidazolinone, and 2,4-dichlorophenoxyacetate (2,4-D).

[0071] In some embodiments, the expression cassette or vector comprising a sequence encoding an RGN polypeptide may further comprise a sequence encoding a crRNA and / or a tracrRNA, or a crRNA and a tracrRNA combined to generate a guide RNA. The sequence encoding the crRNA and / or the tracrRNA may be operably linked to at least one transcription control sequence for expression of the crRNA and / or the tracrRNA in an organism or host cell of interest. For example, the polynucleotide encoding the crRNA and / or the tracrRNA may be operably linked to a promoter sequence recognized by RNA polymerase III (PolIII). Examples of suitable PolIII promoters include, but are not limited to, mammalian U6, U3, H1, and 7SL RNA promoters, and rice U6 and U3 promoters.

[0072] As shown, an expression construct comprising a nucleotide sequence encoding RGN, crRNA, tracrRNA, and / or sgRNA can be used to transform an organism of interest. Methods for transformation include introducing a nucleotide construct into the organism of interest. By "introducing" it is intended to introduce a nucleotide construct into a host cell such that the construct gains access to the interior of the host cell. The methods of the present invention do not require a particular method for introducing a nucleotide construct into a host organism, only for the nucleotide construct to gain access to the interior of at least one cell of the host organism. The host cell can be a eukaryotic or prokaryotic cell. In certain embodiments, the eukaryotic host cell is a plant cell, a mammalian cell, or an insect cell. Methods for introducing a nucleotide construct into plant and other host cells are known in the art, including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.

[0073] This method results in transformed organisms, including plants, including whole plants, as well as plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, propagations, embryos and their progeny. Plant cells can be differentiated or undifferentiated (e.g., calluses, suspension culture cells, protoplasts, leaf cells, root cells, phloem cells, pollen).

[0074] "Transgenic organism" or "transformed organism" or "stably transformed" organism or cell or tissue refers to an organism that incorporates or has incorporated a polynucleotide encoding the RGN, crRNA, and / or tracrRNA of the present invention. It is recognized that other exogenous or endogenous nucleic acid sequences or DNA fragments can also be incorporated into the host cell. Agrobacterium and biolistic mediated transformation are the two approaches primarily used for transformation of plant cells. However, transformation of host cells can be performed by infection, transfection, microinjection, electroporation, microprojection, biolistics or particle gun methods, electroporation, silica / carbon fiber, ultrasound mediated, PEG mediated, calcium phosphate co-precipitation, polycation DMSO technology, DEAE, dextran procedures, and virus mediated, liposome mediated, etc. Viral mediated introduction of polynucleotides encoding the RGN, crRNA, and / or tracrRNA includes retrovirus, lentivirus, adenovirus, and adeno-associated virus mediated introduction and expression, as well as the use of caulimovirus, geminivirus, and RNA plant viruses.

[0075] Transformation protocols as well as protocols for introducing polypeptide or polynucleotide sequences into plants can vary depending on the type of host cell (e.g., monocot or dicotyledonous plant cell) targeted for transformation. Transformation methods are known in the art and include those described in U.S. Patent Nos. 8,575,425, 7,692,068, 8,802,934, and 7,541,517. 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, GW (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 57:3737-3746; Zupan and Zambryski (1995) Plant Physiology 107:1041-1047; Jones et al. (2005) Plant Methods 1:5.

[0076] Transformation can result in stable or transient incorporation of a nucleic acid into a cell. "Stable transformation" means that a nucleotide construct introduced into a host cell is integrated into the genome of the host cell and can be inherited by its progeny. "Transient transformation" means that a polynucleotide is introduced into a host cell and does not integrate into the genome of the host cell.

[0077] 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. This method relies on particle gun delivery of DNA containing a selection marker and targeting the DNA to the plastid genome via homologous recombination. Additionally, plastid transformation can be achieved by transactivation of silent plastid-borne transgenes to express nuclear-encoded and plastid-directed RNA polymerases that are tissue-preferred. Such a system is reported in McBride et al. (1994) Proc. Natl. Acad. Sci. USA 91:7301-7305.

[0078] The transformed cells can be grown into transgenic organisms such as plants according to conventional methods. See, for example, McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants are then grown and pollinated with the same transformed line or a different line, and the resulting hybrids identified for constitutive expression of the desired phenotypic trait. To ensure that the expression of the desired phenotypic trait is stably maintained and inherited, two or more generations can be grown, and then harvested seeds can be obtained to ensure that the expression of the desired phenotypic trait is achieved. In this way, the present invention provides transformed seeds (also called "transgenic seeds") carrying the nucleotide constructs of the present invention, e.g., the expression cassettes of the present invention, stably integrated into their genome.

[0079] Alternatively, transformed cells may be introduced into an organism. These cells may be derived from an organism, where the cells are transformed in an ex vivo approach.

[0080] The sequences provided herein can be used to transform any plant species, including, but not limited to, monocotyledonous and dicotyledonous plants. Examples of plants of interest include, but are not limited to, maize (corn), sorghum, wheat, sunflower, tomato, cruciferous, pepper, potato, cotton, rice, soybean, sugarcane, sweet potato, tobacco, barley, and oilseed rape, brassica, alfalfa, rye, millet, safflower, peanut, sweet potato, cassava, coffee, coconut, pineapple, citrus, cocoa, tea, banana, avocado, fig, guava, mango, olive, papaya, cashew, macadamia, almond, oat, vegetable, ornamental, and conifer.

[0081] Vegetables include, but are not limited to, tomatoes, lettuce, green beans, lima beans, peas, and members of the genus Curcuma such as cucumber, cantaloupe, and musk melon. Ornamental plants include, but are not limited to, azaleas, hydrangeas, hibiscus, roses, tulips, daffodils, petunias, carnations, poinsettias, chrysanthemums, and the like. Preferably, the plants of the present invention are crop plants (e.g., corn, sorghum, wheat, sunflower, tomato, crucifer, pepper, potato, cotton, rice, soybean, sugarcane, tobacco, barley, oilseed rape, and the like).

[0082] As used herein, the term plant includes intact plant cells, plant protoplasts, plant cell tissue cultures capable of regenerating plants, plant callus, plant mass, and plant cells in plants or plant parts (embryos, pollen, ova, seeds, leaves, flowers, branches, fruits, kernels, ears, cobs, husks, stems, roots, root tips, anthers, etc.). Grain means mature seeds produced by commercial producers for purposes other than seed growth or propagation. Progeny, variants, and mutants of regenerated plants are also included within the scope of the invention, provided that these parts contain the introduced polynucleotide. Additionally, processed plant products or by-products, including, for example, soybeans, carrying the sequences disclosed herein are provided.

[0083] Polynucleotides encoding RNA, crRNA, and / or tracrRNA can also 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.).

[0084] Polynucleotides encoding RGN, crRNA, and / or tracrRNA can be used to transform any eukaryotic species, including, but not limited to, animals (e.g., mammals, insects, fish, birds, and reptiles), fungi, amoebae, algae, and yeast.

[0085] Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acid into mammalian cells or target tissues. Such methods can be used to administer nucleic acid encoding components of CRISPR system to cells in culture or in a host organism. Non-viral vector delivery systems include DNA plasmids, RNA (e.g., transcripts of vectors described herein), naked nucleic acid, and nucleic acid complexed with a delivery vehicle (e.g., liposomes). Viral vector delivery systems include DNA and RNA viruses that have either episomal or integrated genomes after delivery to cells. For reviews 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 See Bohm (eds) (1995); and Yu et al., Gene Therapy 1:13-26 (1994).

[0086] 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 drug-enhanced DNA incorporation. Lipofection is described, for example, in U.S. Patent Nos. 5,049,386, 4,946,787; and 4,897,355, and lipofection reagents are commercially available (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner, WO91 / 17424; WO91 / 16024. Delivery can be to cells (e.g., in vitro or ex vivo administration) or to target tissues (e.g., in vivo administration). The preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to those 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); (See U.S. Patent 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).

[0087] The use of RNA or DNA virus-based systems for delivery of nucleic acids takes advantage of the highly evolved processes for targeting viruses to specific cells in the body and transporting the viral payload to the nucleus. Viral vectors can be administered directly to the patient (in vivo) or can be used to treat cells in vitro and the modified cells can be administered to the patient as needed (ex vivo). Traditional virus-based systems can include retroviral, lentiviral, adenoviral, adeno-associated and herpes simplex virus vectors for gene transfer. Integration into the host genome is possible with retroviral, lentiviral and adeno-associated viral gene transfer methods, often resulting in long-term expression of the inserted transgene. Furthermore, high transduction efficiencies have been observed in many different cell types and target tissues.

[0088] The tropism of retroviruses can be altered by incorporating foreign envelope proteins, expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors that can transduce or infect non-dividing cells and typically produce high viral titers. Thus, the choice of retroviral gene transfer system depends on the target tissue. Retroviral vectors consist of cis-acting long terminal repeats with packaging capacity of up to 6-10 kb of foreign sequence. The minimal cis-acting LTRs are sufficient for vector replication and packaging, which are then used to integrate therapeutic genes into target cells to provide permanent transgene expression. Widely used retroviral vectors include those based on murine leukemia virus (MuLV), gibbon apa leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency 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., Viral. 176:58-59 (1990); Wilson et al., J. Viral. 63:2374-2378 (1989); Miller et al., 1. Viral. 65:2220-2224 (1991); PCT / US94 / 05700)).

[0089] For applications where transient expression is preferred, adenovirus-based systems can be used. Adenovirus-based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. High titers and expression levels have been obtained using such vectors. This vector can be produced in large quantities in a relatively simple system. Adeno-associated virus ("AAV") vectors can also be used to transduce cells with target nucleic acids, for example, in in vitro production of nucleic acids and peptides, and in vivo and ex vivo gene therapy procedures (see, for example, West et al., Virology 160:38-47 (1987); U.S. Patent No. 4,797,368; WO93 / 24641; Katin, Human Gene Therapy 5:793-801 (1994); Muzyczka, 1. Clin. Invest. 94:1351 (1994)). The construction of recombinant AAV vectors has been described in many publications, including US patents, such as US Patent 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 viral particles capable of infecting host cells. Such cells include 293 cells, which package adenovirus, and ΨJ2 cells or PA317 cells, which package retrovirus.

[0090] Viral vectors used in gene therapy are usually produced by creating a cell line that packages a nucleic acid vector into a viral particle. The vector typically contains the minimal viral sequences required for packaging and subsequent integration into the host, with other viral sequences being replaced with an expression cassette of the polynucleotide to be expressed. Missing viral functions are typically supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically only have the ITR sequences from the AAV genome required for packaging and integration into the host genome. The viral DNA is packaged into a cell line that contains a helper plasmid that encodes the other AAV genes, namely rep and cap, but lacks the ITR sequences.

[0091] The cell line can also be infected with adenovirus as a helper. The helper virus promotes the replication of AAV vector and the expression of AAV genes from the helper plasmid. The helper plasmid is not packaged in significant amounts because it lacks ITR sequences. Contamination by adenovirus can be reduced, for example, by heat treatment, to which adenovirus is more sensitive than AAV. Further methods for the delivery of nucleic acid to cells are known to those skilled in the art. For example, see US20003 / 0087817, which is incorporated herein by reference.

[0092] In some embodiments, the host cell is transiently or non-transiently transfected with one or more vectors described herein. In some embodiments, the cell is transfected as it naturally occurs in the subject. In some embodiments, the transfected cell is taken from the subject. In some embodiments, the cell is derived from the subject, e.g., a cell line taken from the subject. 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, MiaPa cells, Panel, PC-3, Tfl, CTLL-2, CIR, Rat6, CVI, RPTE, A1O, T24, 182, A 375, ARH-77, Calul, SW480, SW620, SKOV3, SK-UT, CaCo2, P388Dl, SEM-K2, WEHI-231, HB56, TI B55, 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 epithelium, BALB / 3T3 mouse embryonic fibroblasts, 3T3Swiss, 3T3-Ll, 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-10Tl / 2, C6 / 36, Cal-27, CHO, CHO-7, CHO-IR, CHO-Kl, 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, lY cells, K562 cells, Ku812, KCL22, KGl, KYOl, LNCap, Ma-Mel 1-48, MC-38, MCF-7, MCF-l0A, 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 / PNT2, RenCa, RIN-5F, RMA / RMAS, Saos-2 cells, Sf-9, SkBr3, T2, T-47D, T84, THP1 cell lines, U373, U87, U937, VCaP, Vero cells WM39, WT-49, X63, YAC-1, YAR, and transgenic variants thereof. Cell lines are available from a variety of sources known to those of skill in the art (see, for example, the American Type Culture Collection (ATCC) (Manasas, Va.)).

[0093] In some embodiments, cells transfected with one or more vectors described herein are used to establish new cell lines that contain one or more vector-derived sequences. In some embodiments, cells that are transiently transfected with the components of the CRISPR system described herein (e.g., by transient transfection of one or more vectors or transfection with RNA) and modified through the activity of the CRISPR complex are used to establish new cell lines that contain cells that contain the modifications but lack any other exogenous sequences. In some embodiments, cells that are transiently or non-transiently transfected with one or more vectors described herein, or cell lines derived from such cells, are used to evaluate one or more test compounds.

[0094] In some embodiments, one or more of the vectors described herein are used to generate a non-human transgenic animal or a transgenic plant. In some embodiments, the transgenic animal is a mammal, such as a mouse, a rat, or a rabbit.

[0095] IV. Polypeptide and Polynucleotide Variants and Fragments The present disclosure provides active variants and fragments of naturally occurring (i.e., wild-type) RNA-guided nucleases, the amino acid sequences of which are listed as SEQ ID NOs: 1, 9, 16, 23, 30, 38, 46, 54, 61, 69, 75, 82, 89, 95, 103, 110, 117, 137, or 235, as well as SEQ ID NOs: 2, 10, 17, 24, 31, 39, 47, 55, 62, 70, 76, 83, 84, 85, 86, 87, 88, 90, 95, 103, 110, 117, 137, or 235. , 90, 96, 104, 111, 118, 240, 273, or 287, and active variants and fragments of naturally occurring tracrRNA, e.g., SEQ ID NO: 3, 11, 18, 25, 32, 40, 48, 56, 63, 71, 77, 84, 91, 97, 105, 112, 119, 241, 274, or 286, and polynucleotides encoding same.

[0096] The activity of the variants or fragments can be altered compared to the polynucleotide or polypeptide of interest, but the variants and fragments should retain the functionality of the polynucleotide or polypeptide of interest. For example, the variants or fragments can have increased activity, decreased activity, a different spectrum of activity, or any other change in activity when compared to the polynucleotide or polypeptide of interest.

[0097] Fragments and variants of native RGN polypeptides as disclosed herein will retain sequence-specific RNA-guided DNA binding activity. In certain embodiments, fragments and variants of native RGN polypeptides as disclosed herein will retain nuclease activity (single- or double-stranded).

[0098] Fragments and variants of naturally occurring CRISPR repeats such as those disclosed herein will retain the ability of a portion of the guide RNA (including tracrRNA) to bind and guide an RNA-guided nuclease (complexed with the guide RNA) to a target nucleotide sequence in a sequence-specific manner.

[0099] Naturally occurring fragments and variants of tracrRNA as disclosed herein will retain the ability of a portion of a guide RNA (including a CRISPR RNA) to guide an RNA-guided nuclease (complexed with the guide RNA) to a target nucleotide sequence in a sequence-specific manner.

[0100] The term "fragment" refers to a portion of a polynucleotide or polypeptide sequence of the invention. A "fragment" or "biologically active portion" includes a polynucleotide that contains a sufficient number of consecutive nucleotides to retain biological activity (i.e., when contained within a guide RNA, binds to RGN in a sequence-specific manner and targets a target nucleotide sequence). A "fragment" or "biologically active portion" includes a polypeptide that contains a sufficient number of consecutive amino acid residues to retain biological activity (i.e., when complexed with a guide RNA, binds to a target nucleotide sequence in a sequence-specific manner). Fragments of RGN proteins include those that are shorter than the full-length sequence due to the use of alternative downstream start sites. A biologically active portion of an RGN protein can be, for example, a peptide comprising 10, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050 or more consecutive amino acid residues of SEQ ID NO: 1, 9, 16, 23, 30, 38, 46, 54, 61, 69, 75, 82, 89, 95, 103, 110, 117, 137, or 235. Such biologically active portions can be prepared by recombinant techniques and assessed for sequence-specific RNA-guided DNA binding activity. A biologically active fragment of a CRISPR repeat sequence can comprise at least 8 consecutive amino acids of SEQ ID NO: 2, 10, 17, 24, 31, 39, 47, 55, 62, 70, 76, 83, 90, 96, 104, 111, 118, 240, 273, or 287. A biologically active portion of a CRISPR repeat sequence can be, for example, a polynucleotide that comprises 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive nucleotides of SEQ ID NO: 2, 10, 17, 24, 31, 39, 47, 55, 62, 70, 76, 83, 90, 96, 104, 111, 118, 240, 273, or 287.A biologically active portion of a tracrRNA can be, for example, a polynucleotide that includes 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 or more consecutive nucleotides of SEQ ID NO: 3, 11, 18, 25, 32, 40, 48, 56, 63, 71, 77, 84, 91, 97, 105, 112, 119, 241, 274, or 286.

[0101] In general, "variant" is intended to mean a substantially similar sequence. For polynucleotides, variants include deletions and / or additions of one or more nucleotides at one or more internal sites within the native polynucleotide, and / or substitutions of one or more nucleotides at one or more sites within the native polynucleotide. As used herein, a "native" or "wild-type" polynucleotide or polypeptide includes a naturally occurring nucleotide sequence or amino acid sequence, respectively. For polynucleotides, conservative variants include sequences that, due to the degeneracy of the genetic code, encode the native amino acid sequence of the gene of interest. Naturally occurring allelic variants such as these can be identified using well-known molecular biology techniques, for example, polymerase chain reaction and hybridization techniques as outlined below. Variant polynucleotides also include synthetically derived polynucleotides, such as those generated, for example, by using site-directed mutagenesis, but still encoding a polypeptide or polynucleotide of interest. Generally, variants of a particular polynucleotide disclosed herein have at least about 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 that particular polynucleotide as determined by sequence alignment programs and parameters described elsewhere herein.

[0102] Variants of a particular polynucleotide (i.e., a reference polynucleotide) disclosed herein can also be assessed by comparing the percent sequence identity between the polypeptides encoded by the variant polynucleotide and the reference polynucleotide. The percent sequence identity between any two polypeptides can be calculated using sequence alignment programs and parameters described elsewhere herein. When any given pair of polynucleotides disclosed herein is assessed by comparing the percent sequence identity shared by the two polypeptides they encode, the percent sequence identity between the two encoded polypeptides is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.

[0103] In certain embodiments, the presently disclosed polynucleotides encode an RNA-guided nuclease polynucleotide that 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 identity to the amino acid sequence of SEQ ID NO:1, 9, 16, 23, 30, 38, 46, 54, 61, 69, 75, 82, 89, 95, 103, 110, 117, 137, or 235.

[0104] Biologically active variants of the RGN polypeptides of the invention differ by about 1-15 amino acid residues, about 1-10 amino acid residues, about 6-10 amino acids, as few as 5 amino acids, as few as 4 amino acids, as few as 3 amino acids, as few as 2 amino acids, or as few as 1 amino acid residue. In certain embodiments, the polypeptides can include N- or C-terminal truncations, which can include deletions of at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050 or more amino acids from either the N- or C-terminus of the polypeptide.

[0105] In certain embodiments, the presently disclosed polynucleotides comprise or encode CRISPR repeats comprising a nucleotide 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 identity to the nucleotide sequence set forth as SEQ ID NO: 2, 10, 17, 24, 31, 39, 47, 55, 62, 70, 76, 83, 90, 96, 104, 111, 118, 240, 273, or 287.

[0106] The presently disclosed polynucleotides can comprise or encode a tracrRNA comprising a nucleotide 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 identity to the nucleotide sequence set forth as SEQ ID NO: 3, 11, 18, 25, 32, 40, 48, 56, 63, 71, 77, 84, 91, 97, 105, 112, 119, 241, 274, or 286.

[0107] Biologically active variants of CRISPR repeats or tracrRNA of the invention can differ by about 1-15 nucleotides, about 1-10, about 6-10, as few as 5, as few as 4, as few as 3, as few as 2, or as few as 1 nucleotide. In certain embodiments, the polynucleotide can include a 5' or 3' truncation, which can include a deletion of at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 or more nucleotides from the 5' or 3' end of the polynucleotide.

[0108] It is recognized that modifications can be made to the RGN polypeptide, CRISPR repeats, and tracrRNA provided herein to generate modified proteins and polynucleotides. Human-designed changes can be introduced by applying site-directed mutagenesis techniques. Alternatively, naturally occurring, unknown, or unidentified polynucleotides and / or polypeptides that are structurally and / or functionally related to the sequences disclosed herein can also be identified within the scope of the present invention. Conservative amino acid substitutions can be made in non-conserved regions that do not alter the function of the RGN protein. Alternatively, modifications that improve the activity of RGN can be made.

[0109] Mutant polynucleotides and proteins also include sequences and proteins derived from mutagenic and recombinant techniques such as DNA shuffling. In such procedures, one or more different RGN proteins disclosed herein (e.g., SEQ ID NOs: 1, 9, 16, 23, 30, 38, 46, 54, 61, 69, 75, 82, 89, 95, 103, 110, 117, 137, or 235) are manipulated to create new RGN proteins with desired properties. In this manner, libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides that have substantial sequence identity and contain sequence regions that can be homologously recombined in vitro or in vivo. For example, this method can be used to shuffle sequence motifs encoding domains of interest between the RGN sequences provided herein and other known RGN genes to generate increased K for the enzyme. m Novel genes encoding proteins with improved properties of interest, such as . Such strategies for DNA shuffling are known in the art. See Crameri et al. (1998) Nature 391:288-291; U.S. Patent Nos. 5,605,793 and 5,837,458. A "shuffled" nucleic acid is a nucleic acid generated by a shuffling procedure, such as any of the shuffling procedures described herein. Shuffled nucleic acids are generated by recombining (physically or virtually) two or more nucleic acids (or feature strings), e.g., in an artificial, optionally recursive manner. Generally, one or more screening steps are used in the shuffling step to identify nucleic acids of interest; this screening step can be performed before or after any recombination step. In some (but not all) shuffling embodiments, it is desirable to perform multiple rounds of recombination before selection to increase the diversity of the pool to be screened. The entire process of recombination and selection is optionally repeated recursively. Depending on the context, shuffling can refer to the overall process of recombination and selection, or it can simply refer to the recombination part of the overall process.

[0110] As used herein, "sequence identity" or "identity" in the context of two polynucleotide or polypeptide sequences refers to residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentages of sequence identity are used in the context of proteins, it is recognized that non-identical residue positions often differ by conservative amino acid substitutions, where an amino acid residue is replaced with another amino acid residue that has similar chemical properties (e.g., charge or hydrophobicity), thus not changing the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity". Means for making this adjustment are well known to those of skill in the art. Typically, this involves scoring conservative substitutions as partial mismatches rather than complete mismatches, thereby increasing the percentage sequence identity. Thus, for example, where identical amino acids are given a score of 1 and non-conservative substitutions are given a score of zero, conservative substitutions are given a score between zero and 1. Scoring of conservative substitutions is calculated, for example, as implemented in the program PC / GENE (Intelligenetics, Mountain View, Calif.).

[0111] As used herein, "percentage of sequence identity" refers to a value determined by comparing two optimally aligned sequences over a comparison window, where the portion of the polynucleotide sequence within the comparison window may contain additions or deletions (i.e., gaps) compared to a reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which identical nucleic acid bases or amino acid residues occur in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to yield the percentage of sequence identity.

[0112] Unless otherwise specified, sequence identity / similarity values ​​provided herein refer to values ​​obtained using GAP version 10 with the following parameters: % identity and % similarity for nucleotide sequences using GAP weight 50 and length weight 3, and the nwsgapdna.cmp scoring matrix; % identity and % similarity for amino acid sequences using GAP weight 8 and length weight 2; and the BLOSUM62 scoring matrix; or equivalent programs. By "equivalent program" is intended any sequence comparison program that produces alignments that have identical nucleotide or amino acid residue matches and identical percent sequence identity for the two sequences in question when compared to the corresponding alignment produced by GAP version 10.

[0113] Two sequences are "optimally aligned" if they are aligned for similarity scoring using a defined amino acid substitution matrix (e.g., BLOSUM62), gap existence penalty, and gap extension penalty to reach the highest possible score for the pair of sequences. Amino acid substitution matrices and their use in quantifying similarity between two sequences are well known in the art and are described, for example, in Dayhoff et al. (1978) "A model of evolutionary change in proteins", In "Atlas of Protein Sequence and Structure", Vol. 5, Suppl. 3 (ed. MO Dayhoff), pp. 345-352. Natl. Biomed. Res. Found., Washington, DC and Henikoff et al. (1992) Proc. Natl. Acad. Sci. USA 89:10915-1091. The BLOSUM62 matrix is ​​often used as the default scoring substitution matrix in sequence alignment protocols. A gap existence penalty is imposed for introducing a single amino acid gap into one of the aligned sequences, and a gap extension penalty is imposed for an additional empty amino acid position inserted into an already open gap. The alignment is defined by the amino acid positions in each sequence where the alignment begins and ends, and optionally by inserting a gap or gaps in one or both sequences to reach the highest possible score. Although optimal alignment and scoring can be performed manually, the process is facilitated by the use of computer-implemented alignment algorithms such as, for example, Gapped BLAST 2.0, disclosed in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402 and publicly available at the National Center for Biotechnology Information Website (www.ncbi.nlm.nih.gov).Optimal alignments, including multiple alignments, can be prepared, for example, using PSI-BLAST, available via www.ncbi.nlm.nih.gov and described by Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402.

[0114] For an amino acid sequence that is optimally aligned with a reference sequence, an amino acid residue "corresponds" to the position in the reference sequence with which the residue is paired in the alignment. The "position" is indicated by a number that consecutively identifies each amino acid in the reference sequence based on its position relative to the N-terminus. Due to deletions, insertions, truncations, fusions, etc. that must be considered when determining optimal alignment, the number of amino acid residues in a test sequence determined simply by counting from the N-terminus will generally not be the same as the number of its corresponding position in the reference sequence. For example, if there is a deletion in the aligned test sequence, there will be no amino acid that corresponds to the position in the reference sequence at the site of the deletion. If there is an insertion in the aligned reference sequence, the insertion does not correspond to any amino acid position in the reference sequence. In the case of a truncation or fusion, there may be a stretch of amino acids in the reference sequence or aligned sequence that does not correspond to any amino acid in the corresponding sequence.

[0115] V. Antibodies Also included are antibodies against RGN polypeptides or ribonucleoproteins, including the RGN polypeptides of the present invention (SEQ ID NO: 1, 9, 16, 23, 30, 38, 46, 54, 61, 69, 75, 82, 89, 95, 103, 110, or 117, or active variants or fragments thereof). Methods for producing antibodies are well known in the art (see, for example, Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY; and U.S. Patent No. 4,196,265). These antibodies can be used in kits for the detection and isolation of RGN polypeptides or ribonucleoproteins. Thus, the disclosure provides kits that include an antibody that specifically binds to a polypeptide or ribonucleoprotein described herein, including, for example, a polypeptide having a sequence of SEQ ID NO: 1, 9, 16, 23, 30, 38, 46, 54, 61, 69, 75, 82, 89, 95, 103, 110, or 117.

[0116] VI. SYSTEMS AND RIBONUCLEOPROTEIN COMPLEXES BINDING TO A SEQUENCE OF TARGET AND METHODS FOR SYNTHESIS THEREOF The present disclosure provides a system for binding a target sequence of interest, wherein the system comprises at least one guide RNA or nucleotide sequence encoding the same, and at least one RNA guide nuclease or nucleotide sequence encoding the same. The guide RNA hybridizes to the target sequence of interest and forms a complex with the RGN polypeptide, thereby binding the RGN polypeptide to the target sequence. In some of these embodiments, the RGN comprises SEQ ID NO: 1, 9, 16, 23, 30, 38, 46, 54, 61, 69, 75, 82, 89, 95, 103, 110, 117, 137, or 235, or active variants or fragments thereof. In various embodiments, the guide RNA comprises a CRISPR repeat sequence comprising SEQ ID NO: 2, 10, 17, 24, 31, 39, 47, 55, 62, 70, 76, 83, 90, 96, 104, 111, 118, 240, 273, or 287, or an active variant or fragment thereof. In certain embodiments, the guide RNA comprises a tracrRNA comprising SEQ ID NO: 3, 11, 18, 25, 32, 40, 48, 56, 63, 71, 77, 84, 91, 97, 105, 112, 119, 241, 274, or 286, or an active variant or fragment thereof. The guide RNA of the system may be a single guide RNA or a dual guide RNA. In certain embodiments, the system comprises an RNA-guided nuclease that is heterologous to the guide RNA, where the RGN and the guide RNA are not naturally complexed.

[0117] The system for binding a target sequence of interest provided herein can be a ribonucleoprotein complex that is at least one molecule of RNA bound to at least one protein. The ribonucleoprotein complex provided herein comprises at least one guide RNA as the RNA component and an RNA-guided nuclease as the protein component. Such a ribonucleoprotein complex can be purified from a cell or organism that naturally expresses an RGN polypeptide and has been engineered to express a particular guide RNA specific to a target sequence of interest. Alternatively, the ribonucleoprotein complex can be purified from a cell or organism that has been transformed with a polynucleotide encoding an RGN polypeptide and a guide RNA and cultured under conditions that allow expression of the RGN polypeptide and the guide RNA. Thus, methods are provided for making an RGN polypeptide or an RGN ribonucleoprotein complex. Such methods include culturing a cell that comprises a nucleotide sequence encoding an RGN polypeptide, and in some embodiments, a cell that comprises a nucleotide sequence encoding a guide RNA under conditions in which the RGN polypeptide (and in some embodiments, the guide RNA) is expressed. The RGN polypeptide or RGN ribonucleoprotein can then be purified from a lysate of the cultured cells.

[0118] Methods for purifying RGN polypeptides or RGN ribonucleoprotein complexes from lysates of biological samples are known in the art (e.g., size-exclusion and / or affinity chromatography, 2D-PAGE, HPLC, reverse-phase chromatography, immunoprecipitation). In certain methods, the RGN polypeptide is recombinantly produced and contains purification tags to aid in its purification, including, but not limited to, glutathione-S-transferase (GST), chitin-binding protein (CBP), maltose-binding protein, thioredoxin (TRX), poly(NANP), tandem affinity purification (TAP) tags, myc, AcV5, AU1, AU5, E, ECS, E2, FLAG, HA, nus, Softag 1, Softag 3, Strep, SBP, Glu-Glu, HSV, KT3, S, S1, T7, V5, VSV-G, 6xHis, 10xHis, biotin carboxyl carrier protein (BCCP), and calmodulin. Generally, the labeled RGN polypeptide or RGN ribonucleoprotein complex is purified using immobilized metal affinity chromatography, it will be understood that other forms of chromatography or other similar methods known in the art can be used alone or in combination, including, for example, immunoprecipitation.

[0119] An "isolated" or "purified" polypeptide, or a biologically active portion thereof, is substantially or essentially free from components that normally accompany or interact with the polypeptide as found in its natural environment. Thus, an isolated or purified polypeptide is substantially free of other cellular material, or culture medium, if produced by recombinant techniques, or substantially free of chemical precursors or other chemicals, if chemically synthesized. Proteins that are substantially free of cellular material include preparations of the protein having less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating proteins. When the protein of the invention or a biologically active portion thereof is recombinantly produced, optimal culture medium represents less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors or non-protein chemicals of interest.

[0120] Certain methods provided herein for binding and / or cleaving a target sequence of interest include the use of an in vitro assembled RGN ribonucleoprotein complex. In vitro assembly of the RGN ribonucleoprotein complex can be performed using any method known in the art that contacts an RGN polypeptide with a guide RNA under conditions that allow the RGN polypeptide to bind to the guide RNA. As used herein, "contact," "contacting," or "contacting" refers to placing together components of a desired reaction under conditions suitable for carrying out the desired reaction. The RGN polypeptide can be purified from a biological sample, cell lysate, or culture medium, produced by in vitro translation, or chemically synthesized. The guide RNA can be purified from a biological sample, cell lysate, or culture medium, transcribed in vitro, or chemically synthesized. The RGN polypeptide and guide RNA can be contacted in solution (e.g., a buffered saline solution) to allow in vitro assembly of the RGN ribonucleoprotein complex.

[0121] VII. METHODS FOR BINDING, REMOVING OR MODIFYING TARGET SEQUENCES The present disclosure provides a method for binding, cleaving, and / or modifying a target nucleotide sequence of interest. The method comprises delivering a system comprising at least one guide RNA or polynucleotide encoding the same and at least one RGN polypeptide or polynucleotide encoding the same to the target sequence or to a cell, organelle, or embryo that comprises the target sequence. In some of these embodiments, the RGN comprises the amino acid sequence of SEQ ID NO: 1, 9, 16, 23, 30, 38, 46, 54, 61, 69, 75, 82, 89, 95, 103, 110, 117, 137, or 235, or an active variant or fragment thereof. In various embodiments, the guide RNA comprises a CRISPR repeat sequence comprising the nucleotide sequence of SEQ ID NO: 2, 10, 17, 24, 31, 39, 47, 55, 62, 70, 76, 83, 90, 96, 104, 111, 118, 240, 273, or 287, or an active variant or fragment thereof. In certain embodiments, the guide RNA comprises a tracrRNA comprising the nucleotide sequence of SEQ ID NO: 3, 11, 18, 25, 32, 40, 48, 56, 63, 71, 77, 84, 91, 97, 105, 112, 119, 241, 274, or 286, or an active variant or fragment thereof. The guide RNA of the system may be a single guide RNA or a dual guide RNA. The RGN of the system may be a nuclease-dead RGN, may have nickase activity, or may be a fusion polypeptide. In some embodiments, the fusion polypeptide comprises a base-editing polypeptide, such as a cytidine deaminase or an adenosine deaminase. In other embodiments, the RGN fusion protein comprises a reverse transcriptase.In other embodiments, the RGN fusion protein comprises a polypeptide that rescues a member of a functional nucleic acid repair complex, such as a member of the nucleotide excision repair (NER) or transcription-coupled nucleotide excision repair (TC-NER) pathways (Wei et al., 2015, PNAS USA 112(27):E3495-504; Troelstra et al., 1992, Cell 71:939-953; Marnef et al., 2017, J Mol Biol 429(9):1277-1288), as described in U.S. Provisional Application No. 62 / 966,203, filed January 27, 2020, which is incorporated by reference in its entirety. In some embodiments, the RGN fusion protein comprises CSB (van den Boom et al., 2004, J Cell Biol 166(1):27-36; van Gool et al., 1997, EMBO J 16(19):5955-65; an example of which is set forth as SEQ ID NO:268), which is the TC-NER (nucleotide excision repair) pathway and functions as a rescue for other members. In further embodiments, the RNAGNABP fusion protein comprises an active domain of CSB, such as the acidic domain of CSB comprising amino acid residues 356-394 of SEQ ID NO:268 (Teng et al., 2018, Nat Commun 9(1):4115).

[0122] In certain embodiments, the RGN and / or guide RNA are heterologous to the cell, organelle, or embryo into which the RGN and / or guide RNA (or a polynucleotide encoding at least one of the RGN and guide RNA) is introduced.

[0123] In embodiments where the method includes delivering a polynucleotide encoding a guide RNA and / or an RGN polypeptide, the cell or embryo can then be cultured under conditions where the guide RNA and / or the RGN polypeptide is expressed. In various aspects, the method includes contacting the target sequence with an RGN ribonucleoprotein complex. The RGN ribonucleoprotein complex can include an RGN that is nuclease-dead or has nickase activity. In some embodiments, the RGN of the ribonucleoprotein complex is a fusion polypeptide that includes a base-editing polypeptide. In some aspects, the method includes introducing an RGN ribonucleoprotein complex into a cell, organelle, or embryo that includes the target sequence. The RGN ribonucleoprotein complex can be purified from a biological sample, recombinantly produced and then purified, or assembled in vitro as described herein. In embodiments where the RGN ribonucleoprotein complex that contacts the target sequence, organelle, or embryo is assembled in vitro, the method can further include in vitro assembly of the complex prior to contacting the target sequence, cell, organelle, or embryo.

[0124] Purified or in vitro assembled RGN ribonucleoprotein complexes can be introduced into cells, organelles, or embryos using any method known in the art, including, but not limited to, electroporation. Alternatively, RGN polypeptides and / or polynucleotides encoding or comprising guide RNAs can be introduced into cells, organelles, or embryos using any method known in the art (e.g., electroporation).

[0125] Upon delivery or contact with the target sequence, cell, organelle, or embryo containing the target sequence, the guide RNA directs the RGN to bind to the target sequence in a sequence-specific manner. In the embodiment in which the RGN has nuclease activity, the RGN polypeptide cleaves the target sequence of interest upon binding. The target sequence can then be modified through endogenous repair mechanisms, such as non-homologous end joining or homologous repair by the donor polynucleotide provided.

[0126] Methods for measuring the binding of RGN polypeptides to target sequences are known in the art and include chromatin immunoprecipitation assays, gel mobility shift assays, DNA pull-down assays, reporter assays, and microplate capture and detection assays. Similarly, methods for measuring the cleavage or modification of target sequences are known in the art and include in vitro or in vivo cleavage assays in which cleavage is confirmed using PCR, sequencing, or gel electrophoresis, with or without the attachment of an appropriate label (e.g., radioisotope, fluorescent substance) to the target sequence to facilitate detection of degradation products. Alternatively, a nicking-induced exponential amplification reaction (NTEXPAR) assay can be used (see, e.g., Zhang et al. (2016) Chem. Sci. 7:4951-4957). In vivo cleavage can be assessed using the Surveyor assay (Guschin et al. (2010) Methods Mol Biol 649:247-256).

[0127] In some embodiments, the method comprises the use of a single type of RGN complexed with two or more guide RNAs.The multiple guide RNAs can target different regions of one gene or target multiple genes.

[0128] In embodiments where a donor polynucleotide is not provided, the double-stranded break introduced by the RGN polypeptide can be repaired by the non-homologous end joining (NHEJ) repair process. Due to the error-prone nature of NHEJ, the repair of the double-stranded break can result in the modification of the target sequence. As used herein, "modification" refers to a change in the nucleotide sequence of a nucleic acid molecule, which can be the deletion, insertion, or substitution of one or more nucleotides, or a combination thereof. The modification of the target sequence can result in the expression of an altered protein product or the inactivation of the coding sequence.

[0129] In embodiments where a donor polynucleotide is present, the donor sequence in the donor polynucleotide can be integrated or replaced into the target nucleotide sequence during the process of repair of the introduced double-strand break, resulting in the introduction of an exogenous donor sequence. Thus, the donor polynucleotide comprises the donor sequence that is desired to be introduced into the target sequence of interest. In some embodiments, the donor sequence modifies the original target nucleotide sequence so that the newly integrated donor sequence is not recognized and cut by RGN. The integration of the donor sequence can be enhanced by including within the donor polynucleotide a flanking sequence that has substantial sequence identity with the sequence adjacent to the target nucleotide sequence, allowing for a homology-directed repair process. In embodiments where an RGN polypeptide introduces a double-stranded staggered break, the donor polynucleotide comprises a donor sequence flanked by compatible overhangs, allowing for the direct ligation of the donor sequence to the cut target nucleotide sequence containing the overhang by a non-homologous repair process during repair of the double-strand break.

[0130] In embodiments where the method involves the use of an RGN that is a nickase (i.e., that is only capable of cleaving one strand of a double-stranded polynucleotide), the method can include introducing two RGN nickases that target the same or overlapping target sequence and cleave different strands of the polynucleotide. For example, an RGN nickase that cleaves only the positive (+) strand of a double-stranded polynucleotide can be introduced along with a second RGN nickase that cleaves only the negative (-) strand of the double-stranded polynucleotide.

[0131] In various embodiments, a method for binding a target nucleotide sequence and detecting the target sequence is provided, comprising introducing into a cell, organelle, or embryo at least one guide RNA or polynucleotide encoding the same, and at least one RGN polypeptide expressing the guide RNA and / or the RGN polypeptide (if the coding sequence is introduced), wherein the RGN polypeptide is a nuclease-inactive RGN and further comprises a detectable label. The detectable label can be fused to the RGN as a fusion protein (e.g., a fluorescent protein), or can be conjugated to the RGN polypeptide that can be detected visually or by other means, or can be a small molecule incorporated within the RGN polypeptide.

[0132] Also provided herein is a method for regulating the expression of a target sequence or a gene of interest under the regulation of a target sequence. The method comprises introducing into a cell, organelle or embryo at least one guide RNA or polynucleotide encoding the same, and at least one RGN polypeptide or polynucleotide encoding the same, which expresses the guide RNA and / or the RGN polypeptide (when the coding sequence is introduced), wherein the RGN polypeptide is a nuclease-dead RGN. In some of these embodiments, the nuclease-dead RGN is a fusion protein comprising an expression modulator domain (i.e., an epigenetic modification domain, a transcriptional activation domain or a transcriptional repressor domain) as described herein.

[0133] The present disclosure also provides a method for binding and / or modifying a target nucleotide sequence of interest, comprising delivering a system comprising at least one guide RNA or a polynucleotide encoding the same, and at least one fusion polypeptide comprising an RGN and a base-editing polypeptide of the present invention, such as a cytidine deaminase or an adenosine deaminase, or a polynucleotide encoding the fusion polypeptide, to the target sequence or to a cell, organelle, or embryo comprising the target sequence.

[0134] Those skilled in the art will understand that any of the currently disclosed methods can be used to target a single target sequence or multiple target sequences.Thus, the methods include the use of a single RGN polypeptide in combination with multiple separate guide RNAs that can target multiple different sequences within a single gene and / or multiple genes.Also encompassed herein are methods in which multiple different guide RNAs are introduced in combination with multiple different RGN polypeptides.These guide RNAs and guide RNA / RGN polypeptide systems can target multiple different sequences within a single gene and / or multiple genes.

[0135] In one aspect, the present invention provides a kit comprising any one or more of the elements disclosed in the above methods and compositions. In some aspects, the kit comprises a vector system and instructions for using the kit. In some embodiments, the vector system comprises (a) a first regulatory element operably linked to a DNA sequence encoding a crRNA sequence, and one or more insertion sites for inserting a guide sequence upstream of the encoded crRNA sequence, which when expressed directs sequence-specific binding of a CRISPR complex to a target sequence in a eukaryotic cell, and the CRISPR complex comprises a CRISPR enzyme complexed with the guide RNA polynucleotide; and / or (b) an enzyme encoding the CRISPR enzyme comprising a nuclear localization sequence. The elements can be provided individually or in combination and can be provided in any suitable container, such as a vial, bottle, or tube.

[0136] In some embodiments, the kit includes instructions in one or more languages. In some embodiments, the kit includes one or more reagents for use in a process utilizing one or more elements described herein. The reagents can be provided in any suitable container. For example, the kit can provide one or more reaction or storage buffers. The reagents can be provided in a form that can be used in a particular assay or in a form that requires the addition of one or more other components prior to use (e.g., concentrated or lyophilized form). The buffer can be any buffer, including, but not limited to, sodium carbonate buffer, sodium bicarbonate buffer, borate buffer, Tris buffer, MOPS buffer, HEPES buffer, and combinations thereof. In some embodiments, the buffer is alkaline. In some embodiments, the buffer has a pH of about 7 to about 10.

[0137] In some embodiments, the kit comprises one or more oligonucleotides corresponding to the guide sequence for insertion into the vector, so as to operably link the guide sequence and the regulatory element. In some embodiments, the kit comprises a homologous recombination template polynucleotide. In one embodiment, the present invention provides a method of using one or more elements of the CRISPR system. The CRISPR complex of the present invention provides an effective means for modifying a target polynucleotide. The CRISPR complex of the present invention has a wide range of uses, including modifying (e.g., deleting, inserting, moving, inactivating, activating, base editing) a target polynucleotide in multiple cell types. Thus, the CRISPR complex of the present invention has a wide range of applications, for example, in gene therapy, drug screening, disease diagnosis, and prognosis. An exemplary CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized to a target sequence within a target polynucleotide.

[0138] VIII. Target Polynucleotides In one embodiment, the present invention provides a method for modifying a target polynucleotide in a eukaryotic cell, which may be in vivo, ex vivo or in vitro. In one embodiment, the method comprises sampling a cell or a cell population from a human or non-human animal or plant (including microalgae) and modifying the cell or cells. Cultivation may occur at any stage, ex vivo. The cell or cells may be reintroduced into the non-human animal or plant (including microalgae).

[0139] Plant breeders take advantage of natural diversity to combine the most useful genes for desirable properties such as yield, quality, uniformity, resistance, resistance to pests, etc. These desirable properties also include growth, photoperiod preference, temperature requirements, date of onset of floral or reproductive development, fatty acid content, resistance to various environmental factors including insect resistance, disease resistance, nematode resistance, fungal resistance, herbicide resistance, resistance to adverse soil conditions including drought, heat, moisture, cold, wind, and high salinity. Sources of these useful genes include native or exotic varieties, heirloom varieties, wild plant relatives, and induced mutations, e.g., treating plant material with mutagenic agents. With the present invention, plant breeders are provided with a new tool for inducing mutations. Thus, the skilled artisan can analyze genomes for sources of useful genes and use the present invention to induce an increase in useful genes in varieties with desired characteristics or traits, with greater precision than previous mutagens, thus accelerating and improving plant breeding programs.

[0140] The target polynucleotide of the RGN system can be any polynucleotide, endogenous or exogenous to a eukaryotic cell. For example, the target polynucleotide can be a polynucleotide present in the nucleus of a eukaryotic cell. The target polynucleotide can be a sequence that codes for a gene product (e.g., a protein), or a non-coding sequence (e.g., a regulatory polynucleotide or junk DNA). Without wishing to be bound by theory, it is believed that the target sequence should bind to a PAM (protospacer adjacent motif), i.e., a short sequence recognized by the CRISPR complex. The exact sequence and length requirements of the PAM vary depending on the CRISPR enzyme used, but the PAM is typically a 2-5 base pair sequence adjacent to the protospacer (i.e., the target sequence).

[0141] The target polynucleotides of the CRISPR complex may include a number of disease-related genes and polynucleotides, as well as signal transduction biochemical pathway-related genes and polynucleotides. Examples of target polynucleotides include sequences associated with signal transduction biochemical pathways, such as signal transduction biochemical pathway-related genes or polynucleotides. Examples of target polynucleotides include disease-related genes or polynucleotides. A "disease-related" gene or polynucleotide refers to any gene or polynucleotide that produces a transcription or translation product at an abnormal level or in an abnormal form in cells derived from disease-affected tissues compared to non-disease control tissues or cells. This may be a gene that becomes expressed at an abnormally high level or an abnormally low level, whose change in expression correlates with the development and / or progression of the disease. A disease-related gene also refers to a gene that has a mutation or genetic variation that is directly involved or in linkage disequilibrium with a gene involved in the etiology of the disease (e.g., causal mutation). The transcription or translation product may be known or unknown, and may be at a normal or abnormal level. Examples of disease-associated genes and polynucleotides are available from the McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University (Baltimore, Md.) and the National Center for Biotechnology Information, National Library of Medicine (Bethesda, Md.) (available on the World Wide Web).

[0142] Although the CRISPR system is particularly useful due to its relative ease in targeting genomic sequences of interest, the question remains as to what RGN can do to address causative mutations. One approach is to generate a fusion protein between RGN (preferably an inactive or nickase mutant of RGN) and a base editing enzyme or an active domain of a base editing enzyme, such as cytidine deaminase or adenosine deaminase base editing (U.S. Patent No. 9,840,699, incorporated herein by reference). In some embodiments, the method comprises contacting (a) a fusion protein comprising a base editing polypeptide, such as an RGN and a deaminase of the present invention, and (b) a gRNA targeting the fusion protein of (a) with a target nucleotide sequence of a DNA strand, where the DNA molecule is contacted with the fusion protein and the gRNA in an amount and under conditions suitable for deamination of the nucleotide base. In some aspects, the target DNA sequence comprises a sequence associated with a disease or disorder, and deamination of the nucleotide base results in a sequence that is not associated with the disease or disorder. In some embodiments, the target DNA sequence is present in an allele of a crop plant, where a particular allele of a trait of interest results in a plant with reduced agronomic value, and deamination of the nucleotide base results in an allele that improves the trait and increases the agronomic value of the plant.

[0143] In some aspects, the DNA sequence contains a T→C or A→G point mutation associated with a disease or disorder, and deamination of the mutant C or G base results in a sequence that is not associated with the disease or disorder. In some embodiments, deamination corrects the point mutation in the disease or disorder associated sequence.

[0144] In some embodiments, the disease or disorder associated sequence encodes a protein, where deamination introduces a stop codon into the disease or disorder associated sequence, resulting in truncation of the encoded protein. In some embodiments, the contacting is performed in vivo in a subject having, having, or susceptible to being diagnosed with the disease or disorder. In some embodiments, the disease or disorder is a disease associated with a point mutation or single base mutation in the genome. In some embodiments, the disease is a genetic disease, cancer, metabolic disease, or lysosomal storage disease.

[0145] Correcting causal mutations using base editing An example of a genetically inherited disease that can be corrected using the approach that relies on the RGN-base editing fusion protein of the present invention is Hurler syndrome. Hurler syndrome, also known as MPS-1, causes a lysosomal storage disease characterized at the molecular level by the accumulation of dermatan sulfate and heparan sulfate in lysosomes as a result of the deficiency of α-L-iduronidase (IDUA). The disease is generally a genetic disease caused by mutations in the IDUA gene that codes for α-L-iduronidase. Common IDUA mutations are W402X and Q70X, both of which are nonsense mutations that result in premature termination of translation. Such mutations are well addressed by precise genome editing (PGE) approaches, because reversion of a single base, e.g., a base editing approach, restores the wild-type coding sequence, resulting in protein expression controlled by the endogenous regulatory mechanisms of the gene locus. Furthermore, because heterozygotes are known to be asymptomatic, PGE therapy targeting one of these mutations would be useful for the majority of patients with this disease, since only one of the mutated alleles needs to be corrected (Bunge et al. (1994) Hum. Mol. Genet. 3(6): 861-866, incorporated herein by reference).

[0146] Current treatments for Hurler syndrome include enzyme replacement therapy and bone marrow transplantation (Vellodi et al. (1997) Arch. Dis. Child. 76(2): 92-99; Peters et al. (1998) Blood 91(7): 2601-2608, incorporated herein by reference). Enzyme replacement therapy has dramatically impacted the survival and quality of life of Hurler syndrome patients, but this approach requires expensive and time-consuming weekly infusions. Further approaches include delivery of the IDUA gene on an expression vector or insertion of the gene into a highly expressed locus, such as serum albumin (U.S. Pat. No. 9,956,247, incorporated herein by reference). However, these approaches do not allow the restoration of the original IDUA locus to the correct coding sequence. A genome editing strategy would have many advantages. In particular, the regulation of gene expression would be controlled by natural mechanisms present in healthy individuals. Furthermore, base editing does not necessarily cause double-stranded DNA breaks, and may cause large chromosomal rearrangements, cell death, or carcinogenesis due to the disruption of tumor suppression mechanisms. A general strategy may be directed to target and correct specific disease-causing mutations in the human genome using the RGN-base editing fusion proteins of the present invention. It will be understood that similar approaches to target diseases that can be corrected by base editing can also be pursued. It will be further recognized that similar approaches to target disease-causing mutations in other species, particularly common household pets or livestock, can also be deployed using the RGNs of the present invention. Common household pets and livestock include dogs, cats, horses, pigs, cows, sheep, birds, donkeys, snakes, ferrets, fish such as salmon, and shrimp.

[0147] Modification of causative mutations by targeted deletion The RGNs of the present invention may also be useful in human therapeutic approaches where the causative mutations are more complex. For example, some diseases, such as Friedreich ataxia and Huntington's disease, are the result of a significant increase in the repetition of a three-nucleotide motif in a specific region of a gene, affecting the function or expression capacity of the expressed protein. Friedreich ataxia (FRDA) is an autosomal recessive disease that results in progressive degeneration of neural tissue in the spinal cord. Reduced levels of mitochondrial frataxin (FXN) protein cause oxidative damage and iron deficiency at the cellular level. Reduced FXN expression has been associated with the expansion of a GAA triplet within intron 1 of the somatic and germline FXN gene. In FRDA patients, the GAA repeats often consist of more than 70 and sometimes more than 1000 (most commonly 600-900) triplets, whereas unaffected individuals have approximately 40 repeats or less (Pandolfo et al. (2012) Handbook of Clinical Neurology 103: 275-294; Campuzano et al. (1996) Science 271: 1423-1427; Pandolfo (2002) Adv. Exp. Med. Biol. 516: 99-118, all incorporated herein by reference).

[0148] The expansion of the triple repeat sequence that causes Friedreich ataxia (FRDA) occurs at a specific locus in the FXN gene, called the FRDA unstable region. RNA-guided nucleases (RGNs) can be used to excise the unstable region in FRDA patient cells. This approach requires 1) an RGN and a guide RNA sequence that can be programmed to target an allele in the human genome; and 2) a delivery approach for the RGN and guide sequence. Many nucleases used for genome editing, such as the commonly used Cas9 nuclease (SpCas9) from S. pyogenes, are too large to be packaged into an adeno-associated virus (AAV) vector. This makes the approach using SpCas9 more challenging.

[0149] Certain RNA-guided nucleases of the present invention are suitable for packaging with guide RNA into AAV vectors. Although a second vector would be required to pack two guide RNAs, this approach is advantageous compared to that required for larger nucleases such as SpCas9. SpCas9 may require splitting the protein sequence between two vectors. The present invention encompasses a strategy using the RGNs of the present invention in which regions of genomic instability are removed. Such strategies are also applicable to other diseases and disorders with similar genetic basis, such as Huntington's disease. Similar strategies using the RGNs of the present invention are also applicable to similar diseases and disorders in non-human animals of agricultural or economic importance, including dogs, cats, horses, pigs, cows, sheep, birds, donkeys, snakes, ferrets, fish such as salmon, and shrimp.

[0150] Modification of causal mutations by targeted mutagenesis The RGNs of the present invention can also introduce disruptive mutations that confer beneficial effects. Genetic defects in genes encoding hemoglobin, particularly the beta globin chain (HBB gene), can be the cause of many diseases known as hemoglobinopathies, such as sickle cell anemia and thalassemia.

[0151] In adult humans, hemoglobin is a heterotetramer consisting of two alpha (α)-like globin chains and two beta (β)-like globin chains and four heme groups. In adults, the α2β2 tetramer is called hemoglobin A (HbA) or adult hemoglobin. Typically, the α and β globin chains are synthesized in a ratio of about 1:1, which appears to be important for the stabilization of hemoglobin and red blood cells (RBCs). In the developing fetus, another form of hemoglobin, fetal hemoglobin (HbF), is produced, which has a higher binding affinity for oxygen than hemoglobin A and can deliver oxygen to the fetal system via the maternal bloodstream. Fetal hemoglobin also contains two α globin chains, but has two fetal γ globin chains (i.e., fetal hemoglobin is α2γ2) instead of adult β globin chains. The regulation of the switch from the production of γ to β globin is highly complex and primarily involves downregulation of the transcription of γ globin with concomitant upregulation of the transcription of β globin. At approximately 30 weeks of gestation, the synthesis of gamma globin in the fetus begins to decline, whereas the production of β globin increases. By approximately 10 months of age, neonatal hemoglobin is almost all α2β2, although some HbF persists into adulthood (approximately 1–3% of total hemoglobin). In the majority of patients with hemoglobinopathies, the gene encoding gamma globin is present, but expression is relatively low, due to the normal gene repression that occurs around the time of delivery as described above.

[0152] Sickle cell disease is caused by the V6E mutation (GAG to GTG at the DNA level) in the beta-globin gene (HBB), the resulting hemoglobin is called "hemoglobin S" or "HbS", and under hypoxic conditions, HbS molecules aggregate and form fibrillar precipitates. These aggregates cause abnormalities or "sickling" of red blood cells, resulting in a loss of cellular flexibility. Sickle cells can no longer squeeze into capillary beds, which can lead to vaso-occlusive crises in sickle cell patients. Furthermore, sickle cells are more fragile than normal red blood cells and are more prone to hemolysis, ultimately causing anemia in the patient.

[0153] Treatment and management of patients with sickle cell disease is a lifelong proposition that includes antibiotic therapy, pain management, and blood transfusions during acute episodes. One approach is the use of hydroxyurea, which exerts its effects in part by increasing the production of gamma globin. However, the long-term side effects of chronic hydroxyurea therapy are still unknown, and the treatment may result in unwanted side effects and have variable efficacy from patient to patient. Despite the increasing effectiveness of sickle cell disease treatments, the life expectancy of patients is only in the mid-to-late 50s, and morbidity associated with the disease significantly impacts patients' quality of life.

[0154] Thalassemia (α-thalassemia and β-thalassemia) are also hemoglobin-related disorders, typically involving reduced expression of globin chains. This can occur through mutations in the regulatory regions of the genes, or through mutations in the globin coding sequence that result in reduced or decreased levels of expression or functional globin proteins. Treatment of thalassemia usually involves blood transfusions and iron chelation therapy. Bone marrow transplantation has also been used to treat patients with thalassemia major, but carries significant risks if a suitable donor can be identified.

[0155] One approach proposed for the treatment of SCD and β-thalassemia is to increase the expression of gamma globin so that HbF functionally replaces abnormal adult hemoglobin. As mentioned above, the successful treatment of SCD patients with hydroxyurea is believed to be due in part to its effect on increasing gamma globin expression (DeSimone (1982) Proc Nat'l Acad Sci USA 79(14):4428-31; Ley, et al., (1982) N. Engl. J. Medicine, 307: 1469-1475; Ley, et al., (1983) Blood 62: 370-380; Constantoulakis et al., (1988) Blood 72(6):1961-1967, all of which are incorporated herein by reference). Increasing the expression of HbF requires the identification of a gene whose product is involved in the regulation of gamma globin expression. One such gene is BCL11A. BCL11A encodes a zinc finger protein expressed in adult erythroid progenitor cells, and downregulation of its expression leads to increased gamma globin expression (Sankaran et al. (2008) Science 322: 1839, incorporated herein by reference). Although the use of inhibitory RNA targeting the BCL11A gene has been proposed (e.g., U.S. Patent Publication No. 2011 / 0182867, incorporated herein by reference), this technique has several potential drawbacks, including the possibility that complete knockdown may not be achieved, delivery of such RNA may be problematic, and the RNA must be continuously present, requiring multiple treatments over a lifetime.

[0156] The RGNs of the present invention can be used to target the BCL11A enhancer region to disrupt the expression of BCL11A, thereby increasing the expression of gamma globin. This targeted disruption can be achieved by non-homologous end joining (NHEJ), whereby the RGNs of the present invention target specific sequences within the BCL11A enhancer region, creating a double-stranded break, and the cellular machinery repairs the break, typically introducing a deleterious mutation at the same time. Similar to those described for other disease targets, the RGNs of the present invention may have advantages over other known RGNs due to their relatively small size, which allows the expression cassettes for the RGN and its guide RNA to be packaged into a single AAV vector for in vivo delivery. Similar strategies using the RGNs of the present invention are also applicable to similar diseases and disorders in both humans and agriculturally or economically important non-human animals.

[0157] IX. CELLS CONTAINING GENETIC MODIFICATIONS OF POLYNUCLEOTIDES Provided herein are cells and organisms comprising a target sequence of interest modified using the processes mediated by RGN, crRNA, and / or tracrRNA as described herein. In some of these embodiments, the RGN comprises an amino acid sequence of SEQ ID NO: 1, 9, 16, 23, 30, 38, 46, 54, 61, 69, 75, 82, 89, 95, 103, 110, 117, 137, or 235, or an active variant or fragment thereof. In various embodiments, the guide RNA comprises a CRISPR repeat sequence comprising a nucleotide sequence of SEQ ID NO: 2, 10, 17, 24, 31, 39, 47, 55, 62, 70, 76, 83, 90, 96, 104, 111, 118, 240, 273, or 287, or an active variant or fragment thereof. In certain embodiments, the guide RNA comprises a tracrRNA comprising the nucleotide sequence of SEQ ID NO: 3, 11, 18, 25, 32, 40, 48, 56, 63, 71, 77, 84, 91, 97, 105, 112, 119, 241, 274, or 286, or an active variant or fragment thereof. The guide RNA of the system may be a single guide RNA or a dual guide RNA.

[0158] The modified cells can be eukaryotic (e.g., mammalian, plant, insect) or prokaryotic cells. Also provided are organelles and embryos comprising at least one nucleotide sequence modified by the methods utilizing RGN, crRNA, and / or tracrRNA as described herein. The genetically engineered cells, organisms, organelles, embryos can be heterozygous or homozygous for the modified nucleotide sequence.

[0159] Chromosomal modifications of cells, organisms, organelles, or embryos can result in altered expression (up- or down-regulation), inactivation, or expression of modified protein products, or integrated sequences. If the chromosomal modification results in either gene inactivation or expression of a non-functional protein product, the genetically modified cell, organism, organelle, or embryo is called a "knockout." The knockout phenotype can be the result of a deletion mutation (i.e., deletion of at least one nucleotide), an insertion mutation (i.e., insertion of at least one nucleotide), or a nonsense mutation (i.e., substitution of at least one nucleotide such that a stop codon is introduced).

[0160] Alternatively, chromosomal modification of a cell, organism, organelle, or embryo can produce a "knock-in" resulting from chromosomal integration of a nucleotide sequence encoding a protein. In some of these embodiments, the coding sequence is integrated into the chromosome such that the exogenously introduced protein is expressed, while the chromosomal sequence encoding the wild-type protein is inactivated.

[0161] In other embodiments, the chromosomal modification results in the production of a mutant protein product. The expressed mutant protein product can have at least one amino acid substitution and / or at least one amino acid addition or deletion. The modified protein product encoded by the modified chromosomal sequence can exhibit modified characteristics or activities when compared to the wild-type protein, including, but not limited to, modified enzymatic activity or substrate specificity.

[0162] In yet other embodiments, chromosomal modifications can result in altered expression patterns of proteins. As a non-limiting example, chromosomal changes in regulatory regions controlling expression of a protein product can result in overexpression or downregulation of the protein product, or altered tissue or temporal expression patterns.

[0163] X. Kits and methods for detecting target DNA or single-stranded DNA Some RGNs (e.g., APG09106.1 and APG09748, shown as SEQ ID NOs: 54 and 137) can indiscriminately cleave non-target single-stranded DNA (ssDNA) once activated by detection of target DNA. Thus, provided herein are compositions and methods for detecting target DNA (double-stranded or single-stranded) in a sample. In some embodiments, the desired target may exist as RNA, e.g., a genome, or part of the genome of an RNA virus, e.g., a coronavirus. In an embodiment, the coronavirus may be a SARS-like coronavirus. In a further embodiment, the coronavirus may be SARS-CoV-2, SARS-CoV, or a bat SARS-like coronavirus, e.g., bat-SL-CoVZC45 (accession MG772933). In embodiments where the target exists as RNA, the target may be reverse transcribed into a DNA molecule that can be effectively targeted by the RGN. Reverse transcription may be followed by an amplification step such as RT-PCR, which involves thermal cycling, as known in the art, or isothermal methods such as RT-LAMP (reverse transcription loop-mediated isothermal amplification) (Notomi et al., Nucleic Acids Res 28: E63, (2000)).

[0164] These compositions and methods include the use of a detection ssDNA that does not hybridize with the guide RNA and is a non-target ssDNA. In some embodiments, the detection ssDNA includes a detectable label that provides a detectable signal after cleavage of the detection ssDNA. A non-limiting example is a detection ssDNA that includes a fluorophore / quencher pair, where the detection ssDNA does not fluoresce when it is whole (i.e., not left behind) as its signal is suppressed by the presence of the quencher in close proximity. Cleavage of the detection ssDNA results in removal of the quencher, and the fluorescent label can then be detected. Non-limiting examples of fluorescent labels or dyes include Cy5, fluorescein (e.g., FAM, 6 FAM, 5(6)FAM, FITC), Cy3, Alexa Fluor® dyes, and Texas Red. Non-limiting examples of quenchers include Iowa Black® FQ, Iowa Black® RQ, Qx1 quencher, ATT0 quencher, and QSY dyes. In some embodiments, the detection ssDNA can include a second quencher, such as an internal quencher like ZEN™, TAO™, and Black Hole Quencher®, to reduce background and increase signal detection.

[0165] In other embodiments, the detection ssDNA includes a detectable label that provides a detectable signal before cleavage of the detection ssDNA and cleavage of the ssDNA inhibits or prevents detection of the signal. A non-limiting example of such a scenario is the detection ssDNA that includes a fluorescence resonance energy transfer (FRET) pair. FRET is a process in which non-radiative transfer of energy from the excited state of a first (donor) fluorophore to a second (acceptor) fluorophore occurs in close proximity. The emission spectrum of the donor fluorophore overlaps with the excitation spectrum of the acceptor fluorophore. Thus, the acceptor fluorophore will no longer fluoresce when the detection ssDNA is whole (i.e., uncleaved) and the acceptor fluorophore will no longer fluoresce when the detection ssDNA is cleaved because the donor and acceptor fluorophores are no longer in close proximity to each other. FRET donor and acceptor fluorophores are known in the art and include, but are not limited to, cyan fluorescent protein / green fluorescent protein (GFP), Cy3 / Cy5, and GFP / yellow fluorescent protein (YFP).

[0166] In some embodiments, the detector ssDNA has a length of about 2 nucleotides to about 30 nucleotides, including but not limited to, about 2, about 3, about 4, about 5, about 6, about 7, 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 nucleotides, about 26 nucleotides, about 27 nucleotides, about 28 nucleotides, about 29 nucleotides, and about 30 nucleotides.

[0167] A method for detecting a target DNA of a DNA molecule includes contacting a sample with an RGN, contacting the sample with a guide RNA capable of hybridizing with a target DNA sequence in the DNA molecule and a detection single-stranded DNA that does not hybridize with the guide RNA, and then measuring a detectable signal generated by cleavage of the ssDNA by the RGN, thereby detecting the target DNA sequence of the DNA molecule. In some embodiments, the method can include an amplification step of the nucleic acid molecule in the sample before or simultaneously with contacting with the RGN and the guide RNA. In some of these embodiments, the specific sequence to which the guide RNA hybridizes can be amplified to increase the sensitivity of the detection method.

[0168] Samples in which target DNA may be detected using these compositions, and methods involving detecting ssDNA, include any sample that contains or is believed to contain nucleic acid (e.g., DNA or RNA molecules). Samples can be from any source, including synthetic combinations of purified nucleic acid, or biological samples such as respiratory swabs (e.g., nasopharyngeal swabs), cell lysates, patient samples, cells, tissues, saliva, blood, serum, plasma, urine, aspirates, biopsy samples, cerebrospinal fluid, or organisms (bacteria, viruses, etc.).

[0169] The contact of the sample with the RGN, guide RNA, and detection ssDNA can include contact in vitro, ex vivo, or in vivo. In some embodiments, the detection ssDNA and / or RGN and / or guide RNA are immobilized, for example, on a lateral flow device, where the sample contacts the immobilized detection ssDNA and / or RGN and / or guide RNA. In some embodiments, an antibody against an antigen moiety on the detection ssDNA is immobilized, for example, on a lateral flow device, in a manner that allows for the distinction between cleaved detection ssDNA and intact detection ssDNA.

[0170] In some embodiments, the method can further include determining the amount of target DNA present in the sample. The measurement of the detectable signal in the test sample can be compared to a reference measurement (e.g., a measurement of a reference sample or a series thereof containing a known amount of target DNA).

[0171] Non-limiting examples of applications of the compositions and methods include single nucleotide polymorphism (SNP) detection, cancer screening, bacterial infection detection, antibiotic resistance detection, and viral infection detection.

[0172] The detectable signal generated by cleavage of ssDNA by RGN can be measured using any suitable method known in the art, including, but not limited to, measurement of a fluorescent signal, visual analysis of a band on a gel, a colorimetric change, and the presence or absence of an electrical signal.

[0173] The present invention provides a kit for detecting a target DNA of a DNA molecule in a sample, the kit comprising an RGN polypeptide, a guide RNA capable of hybridizing to the RGN, and a target DNA sequence in the DNA molecule, and a detection ssDNA that does not hybridize to the guide RNA.

[0174] Also provided herein is a method of cleaving single stranded DNA by contacting a population of nucleic acids, wherein the population comprises a target DNA sequence of a DNA molecule and a plurality of non-target ssDNAs, with an RGN and a guide RNA capable of hybridizing to the RGN and the target DNA sequence.

[0175] The articles "a" and "an" are used herein to refer to one or more (i.e., at least one) of the grammatical object of the article. By way of example, "a polypeptide" means one or more polypeptides.

[0176] All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this disclosure pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0177] Although the foregoing invention has been described in some detail by way of illustration and example, for purposes of clarity of understanding, it will be apparent that certain changes and modifications can be practiced within the scope of the accompanying embodiments.

[0178] Non-limiting embodiments include: 1. A nucleic acid molecule comprising a polynucleotide encoding an RNA-guided nuclease (RGN) polynucleotide, wherein the polynucleotide comprises a nucleotide sequence encoding an RGN polypeptide comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1, 9, 16, 23, 30, 38, 46, 54, 61, 69, 75, 82, 89, 95, 103, 110, or 117; said RGN polypeptide is capable of binding to a target DNA sequence of a DNA molecule in an RNA guide sequence-specific manner when bound to a guide RNA (gRNA) capable of hybridizing to said target DNA sequence; The nucleic acid molecule, wherein the polynucleotide encoding an RGN polypeptide is operably linked to a promoter heterologous to the polynucleotide. 2. The nucleic acid molecule of embodiment 1, wherein the RGN polypeptide is capable of cleaving the target DNA sequence upon binding. 3. The nucleic acid molecule of embodiment 2, wherein the RGN polypeptide is capable of generating a double-stranded break. 4. The nucleic acid molecule of embodiment 2, wherein the RGN polypeptide is capable of generating a single-strand break. 5. The nucleic acid molecule of embodiment 2, wherein the RGN polypeptide is nuclease inactive.

[0179] 6. A nucleic acid molecule according to any one of embodiments 1 to 5, wherein the RGN polypeptide is functionally fused to the base-editing polypeptide. 7. The nucleic acid molecule of embodiment 6, wherein the base-editing polypeptide is a deaminase. 8. The nucleic acid molecule of embodiment 7, wherein the deaminase is a cytidine deaminase or an adenosine deaminase. 9. The nucleic acid molecule of any one of embodiments 1 to 8, wherein the RGN polypeptide comprises one or more nuclear localization signals. 10. The nucleic acid molecule of any one of embodiments 1 to 9, wherein the RGN polypeptide is codon optimized for expression in a eukaryotic cell.

[0180] 11. The nucleic acid molecule of any one of embodiments 1 to 10, wherein the target DNA sequence is located adjacent to a protospacer adjacent motif (PAM). 12. A vector comprising a nucleic acid molecule according to any one of embodiments 1 to 11. 13. The vector of embodiment 12, further comprising at least one nucleotide sequence encoding said gRNA capable of hybridizing to said target DNA sequence. 14. The vector of embodiment 13, wherein the guide RNA comprises a CRISPR RNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 2, 10, 17, 24, 31, 39, 47, 55, 62, 70, 76, 83, 90, 96, 104, 111, or 118. 15. The vector of embodiment 13 or 14, wherein the gRNA comprises a tracrRNA.

[0181] 16. The vector of embodiment 15, wherein tracrRNA has at least 95% sequence identity to SEQ ID NO: 3, 11, 18, 25, 32, 40, 48, 56, 63, 71, 77, 84, 91, 97, 105, 112, or 119. 17. The vector of embodiment 15 or 16, wherein the gRNA is a single guide RNA. 18. The vector of embodiment 15 or 16, wherein the gRNA is a dual guide RNA. 19. A cell comprising a nucleic acid molecule according to any one of embodiments 1 to 11, or a vector according to any one of embodiments 12 to 18. 20. A method for producing an RGN polypeptide, comprising culturing a cell according to embodiment 18 under conditions in which the RGN polypeptide is expressed.

[0182] 21. 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 95% sequence identity to SEQ ID NO: 1, 9, 16, 23, 30, 38, 46, 54, 61, 69, 75, 82, 89, 95, 103, 110, or 117; wherein the RGN polypeptide is capable of binding to a target DNA sequence of a DNA molecule in an RNA guide sequence-specific manner when bound to a guide RNA (gRNA) capable of hybridizing to the target DNA sequence, Culturing the cells under conditions in which the RGN polypeptide is expressed. A method for producing an RGN polypeptide, comprising: 22. The method of embodiment 20 or 21, further comprising purifying the RGN polypeptide. 23. The method of embodiment 20 or 21, wherein the cell further expresses one or more guide RNAs that bind to the RGN polypeptide to form an RGN ribonucleoprotein complex. 24. The method of embodiment 23, further comprising purifying the RGN ribonucleoprotein complex. 25. A nucleic acid molecule comprising a polynucleotide encoding a CRISPR RNA (crRNA), wherein the crRNA comprises a spacer sequence and a CRISPR repeat sequence, and the CRISPR repeat sequence comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 2, 10, 17, 24, 31, 39, 47, 55, 62, 70, 76, 83, 90, 96, 104, 111, or 118; a) the crRNA; and, optionally, b) a trans-activating CRISPR RNA (tracrRNA) capable of hybridizing to said CRISPR repeat sequence of crRNA; a guide RNA comprising: when bound to an RNA-guided nuclease (RGN) polypeptide, capable of hybridizing to a target DNA sequence of a DNA molecule in a sequence-specific manner via a spacer sequence of said crRNA; The above nucleic acid molecule, wherein the polynucleotide encoding the crRNA is operably linked to a promoter heterologous to the polynucleotide.

[0183] 26. A vector comprising the nucleic acid molecule of embodiment 25. 27. The vector of embodiment 26, wherein the vector further comprises a polynucleotide encoding the tracrRNA. 28. The vector of embodiment 27, wherein the tracrRNA comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 3, 11, 18, 25, 32, 40, 48, 56, 63, 71, 77, 84, 91, 97, 105, 112, or 119. 29. The vector of embodiment 27 or 28, wherein the polynucleotide encoding the crRNA and the polynucleotide encoding the tracrRNA are operably linked to the same promoter and encoded as a single guide RNA. 30. The vector of embodiment 27 or 28, wherein the polynucleotide encoding the crRNA and the polynucleotide encoding the tracrRNA are operably linked to separate promoters.

[0184] 31. The vector of any one of embodiments 26 to 30, wherein the vector further comprises a polynucleotide encoding the RGN polypeptide, and the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1, 9, 16, 23, 30, 38, 46, 54, 61, 69, 75, 82, 89, 95, 103, 110, or 117. 32. A nucleic acid molecule comprising a polynucleotide encoding a trans-activating CRISPR RNA (tracrRNA) comprising a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 3, 11, 18, 25, 32, 40, 48, 56, 63, 71, 77, 84, 91, 97, 105, 112, or 119, a) the tracrRNA; and b) a crRNA comprising a spacer sequence and a CRISPR repeat sequence, the crRNA being capable of hybridizing with the CRISPR repeat sequence of the crRNA; a guide RNA comprising: when bound to an RNA-guided nuclease (RGN) polypeptide, capable of hybridizing to a target DNA sequence of a DNA molecule in a sequence-specific manner via a spacer sequence of said crRNA; The above nucleic acid molecule, wherein the polynucleotide encoding tracrRNA is operably linked to a promoter heterologous to the polynucleotide. The nucleic acid molecule comprising: 33. A vector comprising the nucleic acid molecule of embodiment 32. 34. The vector of embodiment 33, wherein the vector further comprises a polynucleotide encoding the crRNA. 35. The vector of embodiment 34, wherein the CRISPR repeat sequence of the crRNA comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 2, 10, 17, 24, 31, 39, 47, 55, 62, 70, 76, 83, 90, 96, 104, 111, or 118.

[0185] 36. The vector of embodiment 34 or 35, wherein the polynucleotide encoding the crRNA and the polynucleotide encoding the tracrRNA are operably linked to the same promoter and encoded as a single guide RNA. 37. The vector of embodiment 34 or 35, wherein the polynucleotide encoding the crRNA and the polynucleotide encoding the tracrRNA are operably linked to separate promoters. 38. The vector according to any one of embodiments 33 to 37, wherein the vector further comprises a polynucleotide encoding the RGN polypeptide, and the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1, 9, 16, 23, 30, 38, 46, 54, 61, 69, 75, 82, 89, 95, 103, 110 or 117. 39. A system for binding a target DNA sequence of a DNA molecule, the system comprising: a) one or more polynucleotides comprising a nucleotide sequence encoding one or more guide RNAs (gRNAs) or one or more guide RNAs capable of hybridizing to said target DNA sequence; and b) an RNA-guided nuclease (RGN) polypeptide comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1, 9, 16, 23, 30, 38, 46, 54, 61, 69, 75, 82, 89, 95, 103, 110, or 117; or a polynucleotide comprising a nucleotide sequence encoding an RGN polypeptide. Including, the nucleotide sequences encoding one or more guide RNAs and encoding an RNAGN polypeptide are each operably linked to a promoter heterologous to the nucleotide sequence; The above system, wherein one or more guide RNAs are capable of forming a complex with the RGN polypeptide to bind the RGN polypeptide to the target DNA sequence of a DNA molecule. 40. The system of embodiment 39, wherein the gRNA comprises a CRISPR repeat sequence comprising a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 2, 10, 17, 24, 31, 39, 47, 55, 62, 70, 76, 83, 90, 96, 104, 111, or 118.

[0186] 41. The system of embodiment 39 or 40, wherein the gRNA comprises a tracrRNA. 42. The system of embodiment 41, wherein the tracrRNA comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 3, 11, 18, 25, 32, 40, 48, 56, 63, 71, 77, 84, 91, 97, 105, 112, or 119. 43. The system of embodiment 41 or 42, wherein the gRNA is a single guide RNA (sgRNA). 44. The system of embodiment 41 or 42, wherein the gRNA is a dual guide RNA. 45. A system according to any one of embodiments 39 to 44, wherein the target DNA sequence is located adjacent to a protospacer adjacent motif (PAM).

[0187] 46. ​​A system according to any one of embodiments 39 to 45, wherein the target DNA sequence is intracellular. 47. The system of embodiment 46, wherein the cell is a eukaryotic cell. 48. The system of embodiment 47, wherein the eukaryotic cell is a plant cell. 49. The system of embodiment 47, wherein the eukaryotic cell is a mammalian cell. 50. The system of embodiment 47, wherein the eukaryotic cell is an insect cell.

[0188] 51. The system of embodiment 46, wherein the cell is a prokaryotic cell. 52. The system according to any one of embodiments 39 to 51, wherein one or more guide RNAs, when transcribed, can hybridize to a target DNA sequence, and the guide RNAs can form a complex with an RGN polypeptide to directly cleave the target DNA sequence. 53. The system of embodiment 52, wherein the RGN polypeptide is capable of generating a double-stranded break. 54. The system of embodiment 52, wherein the RGN polypeptide is capable of generating a single-strand break. 55. The system of embodiment 52, wherein the RGN polypeptide is nuclease inactive.

[0189] 56. A system described in any one of embodiments 39 to 55, wherein the RGN polypeptide is operably linked to the base-editing polypeptide. 57. The system of embodiment 56, wherein the base-editing polypeptide is a deaminase. 58. The system of embodiment 57, wherein the deaminase is cytidine deaminase or adenosine deaminase. 59. A system according to any one of embodiments 39 to 58, wherein the RGN polypeptide comprises one or more nuclear localization signals. 60. The system of any one of embodiments 39 to 59, wherein the RGN polypeptide is codon optimized for expression in a eukaryotic cell.

[0190] 61. The system according to any one of embodiments 39 to 60, wherein the polynucleotide comprising a nucleotide sequence encoding one or more guide RNAs and the polynucleotide comprising a nucleotide sequence encoding an RGN polypeptide are located on one vector. 62. The system of any one of embodiments 39 to 61, wherein the system further comprises one or more donor polynucleotides or one or more nucleotide sequences encoding one or more donor polynucleotides. 63. A method for binding a target DNA sequence of a DNA molecule, comprising delivering to the target DNA sequence or to a cell containing the target DNA sequence a system according to any one of embodiments 39 to 62. 64. The method of embodiment 63, wherein the RGN polypeptide or the guide RNA further comprises a detectable label, thereby allowing detection of the target DNA sequence. 65. The method of embodiment 63, wherein the guide RNA or the RGN polypeptide further comprises an expression modulator, thereby regulating expression of the target DNA sequence or a gene under transcriptional control by the target DNA sequence.

[0191] 66. A method for cleaving or modifying a target DNA sequence of a DNA molecule, comprising delivering a system described in any one of embodiments 39 to 62 to a target DNA sequence or cell containing the DNA molecule, thereby causing cleavage or modification of the target DNA sequence. 67. The method of embodiment 66, wherein the modified target DNA sequence comprises an insertion of heterologous DNA into the target DNA sequence. 68. The method of embodiment 66, wherein the modified target DNA sequence comprises a deletion of at least one nucleotide from the target DNA sequence. 69. The method of embodiment 66, wherein the modified target DNA sequence comprises a mutation of at least one nucleotide in the target DNA sequence. 70. A method for binding a target DNA sequence of a DNA molecule, the method comprising: a) under conditions suitable for the formation of an RGN-ribonucleotide complex; i) one or more guide RNAs capable of hybridizing to a target DNA sequence; and ii) an RGN polypeptide comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1, 9, 16, 23, 30, 38, 46, 54, 61, 69, 75, 82, 89, 95, 103, 110, or 117; Assembling an RNA-guided nuclease (RGN) ribonucleotide complex in vitro by combining b) contacting a target DNA sequence or a cell containing a target DNA sequence with an in vitro assembled RGN ribonucleotide complex; Including, The above method, wherein one or more guide RNAs hybridize to a target DNA sequence, thereby binding the RGN polypeptide to the target DNA sequence.

[0192] 71. The method according to embodiment 70, wherein the RGN polypeptide or the guide RNA further comprises a detectable label, thereby allowing detection of the target DNA sequence. 72. The method according to embodiment 70, wherein the guide RNA or the RGN polypeptide further comprises an expression modulator, thereby allowing regulation of the expression of the target DNA sequence. 73. A method for cleaving and / or modifying a target DNA sequence of a DNA molecule, comprising: a) an RNA-guided nuclease (RGN) polypeptide, the RGN comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1, 9, 16, 23, 30, 38, 46, 54, 61, 69, 75, 82, 89, 95, 103, 110, or 117; and b) one or more guide RNAs capable of targeting the RGN of (a) to a target DNA sequence; contacting the The above method, wherein one or more guide RNAs hybridize to a target DNA sequence, thereby allowing the RGN polypeptide to bind to the target DNA sequence, resulting in cleavage and / or modification of the target DNA sequence. 74. The method of embodiment 73, wherein cleavage by the RGN polypeptide generates a double-stranded break. 75. The method of embodiment 73, wherein cleavage by the RGN polypeptide generates a single-stranded break.

[0193] 76. The method of embodiment 73, wherein the RGN polypeptide is nuclease inactive. 77. The method of any one of embodiments 73 to 76, wherein the RGN polypeptide is operably linked to the base-editing polypeptide. 78. The method of embodiment 77, wherein the base-editing polypeptide comprises a deaminase. 79. The method of embodiment 78, wherein the deaminase is cytidine deaminase or adenosine deaminase. 80. The method of any one of embodiments 73 to 79, wherein the modified target DNA sequence comprises an insertion of heterologous DNA into the target DNA sequence.

[0194] 81. The method according to any one of embodiments 73 to 79, wherein the modified target DNA sequence comprises a deletion of at least one nucleotide from the target DNA sequence. 82. The method according to any one of embodiments 73 to 79, wherein the modified target DNA sequence comprises a mutation of at least one nucleotide in the target DNA sequence. 83. The method of any one of embodiments 70 to 82, wherein the gRNA comprises a CRISPR repeat sequence comprising a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 2, 10, 17, 24, 31, 39, 47, 55, 62, 70, 76, 83, 90, 96, 104, 111, or 118. 84. The method of any one of embodiments 70 to 83, wherein the gRNA comprises a tracrRNA. 85. The method of embodiment 84, wherein the tracrRNA comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 3, 11, 18, 25, 32, 40, 48, 56, 63, 71, 77, 84, 91, 97, 105, 112, or 119.

[0195] 86. The method of embodiment 84 or 85, wherein the gRNA is a single guide RNA (sgRNA). 87. The method of embodiment 84 or 85, wherein the gRNA is a dual guide RNA. 88. The method of any one of aspects 63 to 87, wherein the target DNA sequence is located adjacent to a protospacer adjacent motif (PAM). 89. The method of any one of embodiments 63 to 88, wherein the target DNA sequence is intracellular. 90. The method of embodiment 89, wherein the cell is a eukaryotic cell.

[0196] 91. The method of embodiment 90, wherein the eukaryotic cell is a plant cell. 92. The method of embodiment 90, wherein the eukaryotic cell is a mammalian cell. 93. The method of embodiment 90, wherein the eukaryotic cell is an insect cell. 94. The method of embodiment 89, wherein the cell is a prokaryotic cell. 95. The method according to any one of embodiments 89 to 94, further comprising culturing cells under conditions in which the RGN polypeptide is expressed and cleaves the target DNA sequence to produce a DNA molecule comprising a modified DNA sequence; and selecting cells comprising the modified target DNA sequence.

[0197] 96. A cell comprising a target DNA sequence modified according to the method of embodiment 95. 97. The cell of embodiment 96, wherein the cell is a eukaryotic cell. 98. The cell of embodiment 97, wherein the eukaryotic cell is a plant cell. 99. A plant comprising a cell according to embodiment 98. 100. A seed comprising a cell according to embodiment 98.

[0198] 101. The cell of embodiment 97, wherein the eukaryotic cell is a mammalian cell. 102. The cell of embodiment 98, wherein the eukaryotic cell is an insect cell. 103. The cell of embodiment 96, wherein the cell is a prokaryotic cell. 104. A method for producing a genetically modified cell that corrects a mutation that causes a genetic disease, the method comprising: a) an RNA-guided nuclease (RGN) polypeptide, the RGN polypeptide comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1, 9, 16, 23, 30, 38, 46, 54, 61, 69, 75, 82, 89, 95, 103, 110, or 117; or a polynucleotide encoding said RGN polypeptide, the polynucleotide encoding the RGN polypeptide being operably linked to a promoter to allow expression of the RGN polypeptide in a cell; and b) a guide RNA (gRNA), wherein the gRNA comprises a CRISPR repeat sequence comprising a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 2, 10, 17, 24, 31, 39, 47, 55, 62, 70, 76, 83, 90, 96, 104, 111, or 118; or a polynucleotide encoding the gRNA, wherein the polynucleotide encoding the gRNA is operably linked to a promoter to allow expression of the gRNA in the cell. Including the introduction of This allows the RGN and gRNA to target the genomic location of the causative mutation and modify the genomic sequence to remove the causative mutation. 105. The method of embodiment 104, wherein the RGN is operably linked to the base-editing polypeptide.

[0199] 106. The method of embodiment 105, wherein the base-editing polypeptide is a deaminase. 107. The method of embodiment 106, wherein the deaminase is cytidine deaminase or adenosine deaminase. 108. The method according to any one of embodiments 104 to 107, wherein the cell is an animal cell. 109. The method of any one of embodiments 104 to 107, wherein the cell is a mammalian cell. 110. The method of embodiment 108, wherein the cells are from a dog, cat, mouse, rat, rabbit, horse, cow, pig, or human.

[0200] 111. The method of embodiment 108, wherein the genetic disease is caused by a single nucleoside polymorphism. 112. The method of embodiment 111, wherein the genetic disease is Haller syndrome. 113. The method of embodiment 112, wherein the gRNA further comprises a spacer sequence that targets a region adjacent to the causative single nucleoside polymorphism. 114. A method for producing a genetically modified cell having a deletion in a disease-causing unstable genomic region, the method comprising: a) an RNA-guided nuclease (RGN) polypeptide, the RGN polypeptide comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1, 9, 16, 23, 30, 38, 46, 54, 61, 69, 75, 82, 89, 95, 103, 110, or 117; or a polynucleotide encoding said RGN polypeptide, the polynucleotide encoding the RGN polypeptide being operably linked to a promoter to allow expression of the RGN polypeptide in a cell; and b) a guide RNA (gRNA), wherein the gRNA comprises a CRISPR repeat sequence comprising a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 2, 10, 17, 24, 31, 39, 47, 55, 62, 70, 76, 83, 90, 96, 104, 111, or 118; or a polynucleotide encoding said gRNA, wherein said polynucleotide encoding the gRNA is operably linked to a promoter to allow expression of the gRNA in the cell, and further wherein the gRNA comprises a spacer sequence that targets the 5' flank of a genomic region of instability; and c) a second guide RNA (gRNA), the gRNA comprising a CRISPR repeat sequence comprising a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 2, 10, 17, 24, 31, 39, 47, 55, 62, 70, 76, 83, 90, 96, 104, 111, or 118; or a polynucleotide encoding the gRNA, the polynucleotide encoding the gRNA being operably linked to a promoter to enable expression of the second gRNA in the cell, and further comprising a spacer sequence that targets the 3' flank of a genomic region of instability. wherein the RGN and the two gRNAs target an unstable genomic region and at least a portion of the unstable genomic region. 115. The method of embodiment 114, wherein the cell is an animal cell.

[0201] 116. The method of embodiment 114, wherein the cell is a mammalian cell. 117. The method of embodiment 115, wherein the cells are from a dog, cat, mouse, rat, rabbit, horse, cow, pig, or human. 118. The method of embodiment 115, wherein the genetically inherited disease is Friedrich's ataxia or Huntington's disease. 119. The method of embodiment 118, wherein the first gRNA further comprises a spacer sequence that targets a region within or near the genomic region of instability. 120. The method of embodiment 118, wherein the second gRNA further comprises a spacer sequence that targets a region within or proximal to the genomic region of instability.

[0202] 121. A method for producing genetically modified mammalian hematopoietic progenitor cells having reduced BCL11A mRNA and protein expression, the method comprising: a) an RNA-guided nuclease (RGN) polypeptide, the RGN polypeptide comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1, 9, 16, 23, 30, 38, 46, 54, 61, 69, 75, 82, 89, 95, 103, 110, or 117; or a polynucleotide encoding said RGN polypeptide, the polynucleotide encoding the RGN polypeptide being operably linked to a promoter to allow expression of the RGN polypeptide in a cell; and b) a guide RNA (gRNA), wherein the gRNA comprises a CRISPR repeat sequence comprising a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 2, 10, 17, 24, 31, 39, 47, 55, 62, 70, 76, 83, 90, 96, 104, 111, or 118; or a polynucleotide encoding the gRNA, wherein the polynucleotide encoding the gRNA is operably linked to a promoter to enable expression of the gRNA in the cell. whereby the RGN and gRNA are expressed in the cells and cleaved at the BCL11A enhancer region, resulting in genetic modification of human hematopoietic progenitor cells and reduced expression of BCL11A mRNA and / or protein. 122. The method of embodiment 121, wherein the gRNA further comprises a spacer sequence that targets a region within or adjacent to the BCL11A enhancer region. 123. The method of any one of embodiments 104 to 122, wherein the guide RNA comprises a tracrRNA comprising a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 3, 11, 18, 25, 32, 40, 48, 56, 63, 71, 77, 84, 91, 97, 105, 112, or 119. 124. A system for binding a target DNA sequence of a DNA molecule, the system comprising: a) one or more polynucleotides comprising one or more nucleotide sequences encoding one or more guide RNAs (gRNAs) or one or more guide RNAs capable of hybridizing to said target DNA sequence; and b) an RNA-guided nuclease (RGN) polypeptide comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1, 9, 16, 23, 30, 38, 46, 54, 61, 69, 75, 82, 89, 95, 103, 110, or 117; Including, wherein the one or more guide RNAs are capable of hybridizing to a target DNA sequence, The above system, wherein one or more guide RNAs are capable of forming a complex with the RGN polypeptide to bind the RGN polypeptide to the target DNA sequence of a DNA molecule. 125. The system of embodiment 124, wherein the RGN polypeptide is nuclease inactive or can function as a nickase.

[0203] 126. The system of embodiment 124 or 125, wherein the RGN polypeptide is functionally fused to a base-editing polypeptide. 127. The system of embodiment 126, wherein the base-editing polypeptide is a deaminase. 128. The system of embodiment 127, wherein the deaminase is cytidine deaminase or adenosine deaminase. 129. A method for detecting a target DNA sequence of a DNA molecule in a sample, comprising: a) The sample i) an RNA-guided nuclease (RGN) polypeptide comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1, 9, 16, 23, 30, 38, 46, 54, 61, 69, 75, 82, 89, 95, 103, 110, 117, or 137, wherein the RGN polypeptide is capable of binding to a target DNA sequence of a DNA molecule in an RNA-guided sequence-specific manner when bound to a guide RNA capable of hybridizing to the target DNA sequence; ii) the guide RNA iii) Detection single-stranded DNA (ssDNA) that does not hybridize with the guide RNA contacting the b) Detection by RGN The above method involves measuring a detectable signal generated by cleavage of ssDNA, thereby detecting the target DNA. 130. The method of embodiment 129, wherein the sample comprises DNA molecules derived from a cell lysate.

[0204] 131. The method of embodiment 129, wherein the sample comprises cells. 132. The method of embodiment 131, wherein the cell is a eukaryotic cell. 133. The method of embodiment 129, wherein the DNA molecule containing the target DNA sequence is generated by reverse transcription of an RNA template molecule present in a sample containing RNA. 134. The method of embodiment 133, wherein the RNA template molecule is an RNA virus. 135. The method of embodiment 134, wherein the RNA virus is a coronavirus.

[0205] 136. The method of embodiment 135, wherein the coronavirus is a bat SARS-like coronavirus, SARS-CoV, or SARS-CoV-2. 137. The method according to any one of embodiments 133 to 136, wherein the sample comprising RNA is derived from a sample comprising cells. 138. The method according to any one of embodiments 129 to 137, wherein the detection ssDNA comprises a fluorophore / quencher pair. 139. The method of any one of embodiments 129 to 137, wherein the detection ssDNA comprises a fluorescence resonance energy transfer (FRET) pair. 140. The method of any one of embodiments 129 to 139, wherein the guide RNA comprises a CRISPR repeat sequence comprising a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 2, 10, 17, 24, 31, 39, 47, 55, 62, 70, 76, 83, 90, 96, 104, 111, 118, or 273.

[0206] 141. The method of any one of embodiments 129 to 140, wherein the guide RNA comprises tracrRNA. 142. The method of embodiment 141, wherein the tracrRNA comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 3, 11, 18, 25, 32, 40, 48, 56, 63, 71, 77, 84, 91, 97, 105, 112, 119, or 274. 143. The method of embodiment 141 or 142, wherein the guide RNA is a single guide RNA. 144. The method of embodiment 141 or 142, wherein the guide RNA is a dual guide RNA. 145. The method of any one of embodiments 129 to 144, wherein the method further comprises amplifying nucleic acid in the sample prior to or together with the contacting of step a.

[0207] 146. The method of embodiment 145, wherein the amplification is a DNA molecule produced by reverse transcription of an RNA molecule. 147. A kit for detecting a target DNA sequence of a DNA molecule in a sample, the kit comprising: a) an RNA-guided nuclease (RGN) polypeptide comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1, 9, 16, 23, 30, 38, 46, 54, 61, 69, 75, 82, 89, 95, 103, 110, 117, or 137, said RGN polypeptide being capable of binding to said target DNA sequence of a DNA molecule in an RNA-guided sequence-specific manner when bound to a guide RNA capable of hybridizing to said target DNA sequence; b) the guide RNA; and c) Detect single-stranded DNA (ssDNA) that does not hybridize with the guide RNA The above kit comprising: 148. The kit according to embodiment 147, wherein the detection ssDNA comprises a fluorophore / quencher pair. 149. The kit of embodiment 147, wherein the detection ssDNA comprises a fluorescence resonance energy transfer (FRET) pair. 150. The kit according to any one of embodiments 147 to 149, wherein the guide RNA comprises a CRISPR repeat sequence comprising a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 2, 10, 17, 24, 31, 39, 47, 55, 62, 70, 76, 83, 90, 96, 104, 111, 118, or 273.

[0208] 151. The kit according to any one of embodiments 147 to 150, wherein the guide RNA comprises tracrRNA. 152. The kit of embodiment 151, wherein the tracrRNA comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 3, 11, 18, 25, 32, 40, 48, 56, 63, 71, 77, 84, 91, 97, 105, 112, 119, or 274. 153. The kit of embodiment 151 or 152, wherein the guide RNA is a single guide RNA. 154. The kit of embodiment 151 or 152, wherein the guide RNA is a dual guide RNA. 155. A method for cleaving single-stranded DNA, the method comprising: subjecting a population of nucleic acids to a DNA molecule containing a target DNA sequence and a plurality of non-target ssDNAs; a) an RNA-guided nuclease (RGN) polypeptide comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1, 9, 16, 23, 30, 38, 46, 54, 61, 69, 75, 82, 89, 95, 103, 110, 117, or 137, wherein the RGN polypeptide is capable of binding to a target DNA sequence in an RNA-guided sequence-specific manner when bound to a guide RNA capable of hybridizing to the target DNA sequence; and b) the guide RNA wherein the RGN polypeptide cleaves the plurality of non-target ssDNA.

[0209] 156. The method of embodiment 155, wherein said population of nucleic acids is in a cell lysate. 157. The method of embodiment 155 or 156, wherein the guide RNA comprises a CRISPR repeat sequence comprising a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 2, 10, 17, 24, 31, 39, 47, 55, 62, 70, 76, 83, 90, 96, 104, 111, 118, or 273. 158. The method of any one of embodiments 155 to 157, wherein the guide RNA comprises tracrRNA. 159. The method of embodiment 158, wherein the tracrRNA comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 3, 11, 18, 25, 32, 40, 48, 56, 63, 71, 77, 84, 91, 97, 105, 112, 119, or 274. 160. The method of embodiment 158 ​​or 159, wherein the guide RNA is a single guide RNA.

[0210] 161. The method of embodiment 158 ​​or 159, wherein the guide RNA is a dual guide RNA. 162. A nucleic acid molecule comprising a polynucleotide encoding a CRISPR RNA (crRNA), wherein the crRNA comprises a spacer sequence and a CRISPR repeat sequence, and the CRISPR repeat sequence comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 240; a) the crRNA; and optionally b) a trans-activating CRISPR RNA (tracrRNA) capable of hybridizing to said CRISPR repeat sequence of crRNA; a guide RNA comprising: when bound to an RNA-guided nuclease (RGN) polypeptide, capable of hybridizing to a target DNA sequence of a DNA molecule in a sequence-specific manner via a spacer sequence of said crRNA; A nucleic acid molecule in which the polynucleotide encoding the crRNA is operably linked to a promoter heterologous to the polynucleotide. 163. A vector comprising a nucleic acid molecule according to embodiment 162. 164. The vector of embodiment 163, wherein the vector further comprises a polynucleotide encoding the tracrRNA. 165. The vector of embodiment 164, wherein the tracrRNA comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 241.

[0211] 166. The vector of embodiment 164 or 165, wherein the polynucleotide encoding the crRNA and the polynucleotide encoding the tracrRNA are operably linked to the same promoter and encoded as a single guide RNA. 167. The vector of embodiment 164 or 165, wherein the polynucleotide encoding the crRNA and the polynucleotide encoding the tracrRNA are operably linked to separate promoters. 168. The vector according to any one of embodiments 163 to 167, wherein the vector further comprises a polynucleotide encoding the RGN polypeptide, the RGN polypeptide comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 235. 169. A nucleic acid molecule comprising a polynucleotide encoding a trans-activating CRISPRRNA (tracrRNA), comprising a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 241, a) the tracrRNA; and b) a crRNA comprising a spacer sequence and a CRISPR repeat sequence, wherein the tracrRNA is capable of hybridizing to the CRISPR repeat sequence of the crRNA; wherein the guide RNA, when bound to an RNA-guided nuclease (RGN) polypeptide, is capable of hybridizing in a sequence-specific manner to a target DNA sequence of a DNA molecule via a spacer sequence of the crRNA, and the polynucleotide encoding the tracrRNA is operably linked to a promoter heterologous to the polynucleotide. 170. A vector comprising a nucleic acid molecule according to embodiment 169.

[0212] 171. The vector described in embodiment 170, wherein the vector further comprises a polynucleotide encoding the crRNA. 172. The vector of embodiment 171, wherein the CRISPR repeat sequence of the crRNA comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 240. 173. The vector of embodiment 171 or 172, wherein the polynucleotide encoding the crRNA and the polynucleotide encoding the tracrRNA are operably linked to the same promoter and encoded as a single guide RNA. 174. The vector of embodiment 171 or 172, wherein the polynucleotide encoding the crRNA and the polynucleotide encoding the tracrRNA are operably linked to separate promoters. 175. The vector described in any one of embodiments 170 to 174, wherein the vector further comprises a polynucleotide encoding the RGN polypeptide, and the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 235.

[0213] The following examples are offered by way of illustration and not by way of limitation. EXAMPLES

[0214] Example 1. Identification of RNA-guided nucleases Seventeen CRISPR-associated RNA-guided nucleases (RGNs) were identified and are listed in Table 1 below. Table 1 shows the name of each RGN, its amino acid sequence, origin, processed crRNA and tracrRNA sequence (see Example 2 for identification method). Table 1 further provides common single guide RNA (sgRNA) sequences, where poly-N indicates the position of the spacer sequence that determines the nucleic acid target sequence of the sgRNA. RGN systems APG05733.1, APG06207.1, APG01647.1, APG08032.1, APG02675.1, APG01405.1, APG06250.1, APG04293.1, and APG01308.1 had a conserved sequence at the base of the hairpin stem of UNA's tracrRNA (SEQ ID NO: 8). In APG05712.1, the sequence at the same position is CNANNG (SEQ ID NO: 37). In APG01658.1, the sequence at the same position is CNANU (SEQ ID NO: 45). For the RGN systems APG06498.1 and APG06877.1, the conserved sequence at the base of the hairpin stem of tracrRNA is UNANG (SEQ ID NO: 53). In APG09882.1 and APG06646.1, the sequence at the same position is UNANC (SEQ ID NO: 68). In APG09053.1, the sequence at the same position is CNANU (SEQ ID NO: 102).

[0215] [Table 1]

[0216] Example 2: Identification of guide RNAs and construction of sgRNAs Cultures of bacteria naturally expressing the RNA-guided nuclease system under study were grown to mid-log phase (OD600 ~ 0.600), pelleted, and flash frozen. RNA was isolated from the pellet using the mirVANA miRNA isolation kit (Life Technologies, Carlsbad, CA) and library preparations were fractionated on a 6% polyacrylamide gel with the NEBNext Small RNA Library Prep kit (NEB, Beverly, MA) to capture RNA species less than 200 nt and detect crRNA and tracrRNA, respectively. Reads were deep sequenced (75 bp paired ends) on a Next Seq 500 (High Output kit) by a service provider (MoGene, St. Louis, MO), quality trimmed using Cutadapt, and mapped to the reference genome using Bowtie2. A custom RNAseq pipeline was created in python to detect crRNA and tracrRNA transcripts. Processed crRNA boundaries were determined by sequence coverage of natural repeat spacer arrays. The anti-repeat portion of the tracrRNA was identified using permissive BLASTn parameters. The boundaries of the processed tracrRNA were confirmed by identifying transcripts containing the anti-repeat from the RNA base sequence depth. Manual curing of the RNA was performed using secondary structure prediction with the RNA folding software NUPACK. sgRNA cassettes were prepared by DNA synthesis and generally designed as follows: (5'->3'): a 20-30 bp spacer operably linked to the processed repeat portion of the crRNA at its 3' end, operably linked to a 4 bp non-complementary linker (AAAG; SEQ ID NO: 123), operably linked to the processed tracrRNA at its 3' end (other 4 bp non-complementary linkers can also be used).

[0217] For in vitro assays, sgRNAs were synthesized by in vitro transcription of sgRNA cassettes using the GeneArt™ Precision gRNA Synthesis Kit (ThermoFisher). The processed crRNA and tracrRNA sequences for each RGN polypeptide were identified and are shown in Table 1. For sgRNAs constructed for PAM libraries 1 and 2, see below.

[0218] Example 3: Determining PAM requirements for each RGN PAM requirements for each RGN were determined using a PAM removal assay essentially adapted from Kleinstiver et al. (2015) Nature 523:481-485 and Zetsche et al. (2015) Cell 163:759-771. Briefly, two plasmid libraries (L1 and L2) were generated in a pUC18 backbone (ampR), each containing a different 30 bp protospacer (target) sequence flanked by eight random nucleotides (i.e., PAM regions). The target sequences and flanking PAM regions of library 1 and library 2 for each RGN are shown in Table 2.

[0219] The libraries were separately electroporated into E. coli BL21(DE3) cells harboring the pRSF-1b expression vector containing the RGN (codon optimized for E. coli) of the invention together with cognate sgRNAs containing spacer sequences corresponding to the L1 or L2 protospacer. 6Enough library plasmid was used to obtain CFU. Both RGN and sgRNA in the pRSF-1b backbone were under the control of a T7 promoter. The transformation reaction was allowed to recover for 1 h before being diluted into LB medium containing carbenicillin and kanamycin and grown overnight. The next day, the mixture was diluted into the autoinducing Overnight Express™ Instant TB Medium (Millipore Sigma) to allow expression of RGN and sgRNA, and after further growth for 4 or 20 h, the cells were centrifuged and the plasmid DNA was purified using a Mini-prep kit (Qiagen, German town, ￥32). In the presence of the appropriate sgRNA, plasmids containing PAMs recognized by RGN are cleaved, resulting in their removal from the population. Plasmids containing PAMs that cannot be recognized by RGN or that are transformed into bacteria without the appropriate sgRNA survive and replicate. The PAM and protospacer regions of the uncleaved plasmid were PCR amplified and cloned according to published protocols (16s-Metagenomic Library Prep Guide 15044223B, Illumina, San Jose, CA). The amplicons were prepared for sequencing according to the ELISA Kit (Illumina, Diego, CA). Deep sequencing (75 bp single-end reads) was performed on a MiSeq (Illumina) by a service provider (MoGene, St. Louis, MO). Typically, 1–4M reads per amplicon were obtained. PAM regions were extracted, counted, and normalized to the total reads for each sample. PAMs leading to plasmid cleavage were identified by their under-representation compared to the control (i.e., when the library was transformed into E. coli containing the RGN but lacking the appropriate sgRNA). To represent the PAM requirement for a novel RGN, the depletion ratio (frequency in the sample / frequency in the control) for all sequences in the region in question was converted to an enrichment value by -log base 2 transformation. Sufficient PAMs were defined as those with an enrichment value >2.3 (which corresponds to a depletion ratio <~0.2). PAMs above this threshold in both libraries were collected and used to generate weblogos, which can be generated, for example, using a web-based service on the Internet known as "weblogos".PAM sequences were identified and reported if there was a consistent pattern in the highest enriched PAMs. The consensus PAMs (enrichment factor (EF) > 2.3) for each RGN are shown in Table 2. The PAM orientation is also shown in Table 2. As described elsewhere in this application, APG06646.1 and APG04293.1 nucleases do not have a PAM interaction domain. The results in Table 2 show that they do not have the typical PAM requirement of 2-5 nucleotides. APG06646.1 and APG04293.1 were shown to have a single base required for cleavage.

[0220] [Table 2]

[0221] Example 4: Engineering guide RNA to enhance nuclease activity 4.1 RGN APG09748 and APG09106.1 For RGN, APG09748 (SEQ ID NO: 137, and the crRNA repeat, tracrRNA, and general sgRNA sequences of APG09748 are set forth as SEQ ID NO: 273, 274, and 275, respectively; all sequences are set forth in International Application No. PCT / US2019 / 068079, incorporated herein by reference), and APG09106.1 have very high sequence identity and have the same PAM, but RNA folding predictions were used to determine regions in the guide RNA that could be modified to optimize nuclease activity. Stability of the crRNA:tracrRNA base pairs in the repeat:anti-repeat region was increased by shortening the repeat:anti region, adding GC base pairs, and removing GU wobble pairs. The "optimized" guide mutants were tested and compared to wild-type gRNA in an in vitro cleavage assay using RGN APG09748.

[0222] To generate RGN for RNP formation, expression plasmids containing RGN fused to a C-terminal His6 (SEQ ID NO: 276) or His10 (SEQ ID NO: 277) tag were constructed and transformed into the BL21(DE3) strain of E. coli. Expression was performed using Magic Media (Thermo Fisher) supplemented with 50 μg / mL kanamycin. After lysis and clarification, proteins were purified by immobilized metal affinity chromatography and quantified using the Qubit Protein Quantitation Kit (Thermo Fisher) or by UV-vis using calculated extinction coefficients.

[0223] Ribonucleoproteins (RNPs) were prepared by incubating purified RGN with sgRNA at approximately a 2:1 ratio for 20 min at room temperature. For in vitro cleavage reactions, RNPs were incubated with plasmids or linear dsDNA containing a target protospacer flanked by preferred PAM sequences for >30 min at room temperature. Two target nucleic acid sequences within the TRAC locus were tested: TRAC11 (SEQ ID NO: 278) and TRAC14 (SEQ ID NO: 279). gRNAs were assayed for target activity both with the correct target nucleic acid sequence (e.g., gRNA has a TRAC11 spacer sequence and the assayed target is TRAC11) and with the correct target nucleic acid sequence (e.g., gRNA has a TRAC11 spacer sequence and the assayed target is TRAC14). Activity, as determined by plasmid cleavage, is assessed by agarose gel electrophoresis. Results are shown in Table 3. Guide variants are listed as SEQ ID NOs: 280-283 and include spacer sequences. These guide sequences use a non-complementary nucleotide linker of AAAA (SEQ ID NO:284). The optimized gRNA (SEQ ID NO:285; poly-N indicates the location of the spacer sequence) with increased repeat:anti-repeat binding has an optimized tracrRNA (SEQ ID NO:286) and an optimized crRNA (SEQ ID NO:287) component. The optimized guide variants were able to cleave two loci where no cleavage was previously detected using wild-type guide RNA. Optimization of hybridization in the repeat:anti-repeat region increased in vitro cleavage of APG09748 from 0% cleavage to 100% cleavage for multiple targets at the TRAC locus.

[0224] [Table 3]

[0225] Further optimized gRNA variants were designed and assayed. In addition, different lengths of the spacer sequence were also tested to determine how the spacer length affects the cleavage efficiency. The sgRNA outside the spacer sequence is referred to as the "backbone" in this assay. In Table 4, these are shown as "WT" (SEQ ID NO: 288, wild-type sequence), and three optimized sgRNAs: V1 (SEQ ID NO: 289), V2 (SEQ ID NO: 290) and V3 (SEQ ID NO: 291). All of these sequences have poly-N to indicate the location of the spacer sequence. The guides were expressed as sgRNAs by in vitro transcription (IVT). Compared to the wild-type sgRNA backbone, V1 is 87.8% identical, V2 is 92.4% identical, and V3 is 85.5% identical. A synthetic tracheal rRNA:crRNA duplex ("synthetic"), which represents a dual guide RNA but is otherwise similar to the wild-type and optimized sgRNAs described above, was also generated and tested.

[0226] For this set of assays, RGN APG09106.1 was used; otherwise, the methods for the in vitro cleavage reactions were similar to those described above. The target nucleic acid sequences were Target 1 (SEQ ID NO:292) and Target 2 (SEQ ID NO:293). The results are shown in Table 4.

[0227] [Table 4]

[0228] 4.2 RGN APG07433.1 RNA folding prediction was used to determine a region in the guide RNA for APG07433.1 (set forth as SEQ ID NO:235 and described in U.S. Patent Applications 2019 / 0367949 and WO2019 / 236566, each of which is incorporated herein by reference in its entirety) that can be modified to optimize nuclease activity and shorten the guide RNA for packaging into viral vectors. Repeat:anti-repeat pairings between the crRNA and tracrRNA regions and the terminal hairpin of the tracrRNA were identified as potential sites for alteration. The repeat:anti-repeat regions were truncated to lengths of 7 (APG07433.1-7bp, SEQ ID NOs:238 and 239), 13 (APG07433.1-13bp, SEQ ID NOs:250 and 251), and 15 base pairs (APG07433.1-15bp, SEQ ID NOs:242 and 243). Additionally, a fourth mutant was tested that altered the sequence of the repeat:anti-repeat region, reducing the pairing to 11 base pairs in length (APG07433.1-11bp-syn, SEQ ID NOs:240 and 241) and introducing a smaller RNA bulge compared to the wild-type guide. Cuts were also made to the terminal hairpins in tracrRNA, including reducing the native sequence to 40 nucleotides (APG07433.1-40nt THP, SEQ ID NOs:254 and 255) and 42 nucleotides (APG07433.1-42nt THP, SEQ ID NOs:244 and 245). Engineered stem-loops were also designed to shorten these to 35 nucleotides (APG07433.1-35ntTHP-syn, SEQ ID NOs:248 and 249) and 39 nucleotides (APG07433.1-39ntTHP-syn, SEQ ID NOs:246 and 247). One guide combined a 13 base pair truncated repeat:antirepeat region with a truncated terminal hairpin of 42 nucleotides (APG07433.1-13bp42ntTH, SEQ ID NOs:252 and 253). The effect of spacer length on cleavage was also examined.Spacer lengths of 25 (APG07433.1-native

[25] , SEQ ID NOs: 236 and 237; spacer sequence shown as SEQ ID NO: 271) and 18 (APG07433.1-native

[18] , SEQ ID NOs: 256 and 257; spacer sequence shown as SEQ ID NO: 272) nucleotides on the wild-type guide RNA backbone were also tested.

[0229] The following dual guide RNAs were prepared by annealing the crRNA and tracrRNA by preparing a solution containing 20 μM crRNA and 10 μM tracrRNA in annealing buffer (Synthego) and then heating to 78° C. for 10 min and then 37° C. for 30 min. RNPs were formed by incubating with purified APG07433.1 protein at 0.5 μM and dual guide RNAs were incubated at 1 μM in phosphate buffered saline for 20 min. The guide RNAs used in this experiment are shown in Table 5.

[0230] [Table 5]

[0231] These RNPs were incubated with PCR products containing the appropriate target sequence (SEQ ID NO: 260) generated by amplification with FAM- and Cy3-labeled primers to facilitate accurate and sensitive quantification of cleavage products. Reactions contained 250 nM RNPs prepared as described above and 150 nM FAM- and Cy3-labeled PCR products in 1× Cutsmart buffer (New England Biolabs). Reactions were allowed to proceed for 15 min at 37° C. and stopped by adding RNase A to 0.1 mg / mL and EDTA to 45 mM. Quenched reactions were heated at 50° C. for 30 min and 95° C. for 5 min. Samples were then analyzed using native acrylamide gel electrophoresis on 5% TBE gels (Bio-rad). They were imaged on a ChemiDoc MP imager. Each of the two dyes could be used for quantification and each sample was run in duplicate. The results of this experiment are shown in Table 6.

[0232] [Table 6]

[0233] This analysis demonstrates that the native backbone outperforms most of the truncated variants and variants containing truncated target sequences (APG07433.1-native

[18] , SEQ ID NO: 256 and 257). Of the truncated backbone sequences, the one with the highest level of truncation is the sequence designated APG07433.1-11bp-syn, which contains a 25nt targeting sequence (spacer; SEQ ID NO: 271), SEQ ID NO: 240 for the crRNA and SEQ ID NO: 241 as the tracrRNA. This guide variation contained a modified repeat:anti-repeat stem loop with an engineered bulge in the stem.

[0234] Example 5: Demonstration of gene editing activity in mammalian cells RGN expression cassettes were generated and introduced into vectors for mammalian expression. RGNs APG09748, APG09106.1, APG05712.1, APG01658.1, APG05733.1, APG06498.1, APG06646.1, APG09882.1, APG01405.1, and APG01308.1 were codon-optimized for mammalian expression (SEQ ID NOs: 304, 305, and 411-418), respectively, and the expressed proteins were operably fused to an SV40 nuclear localization sequence (NLS; SEQ ID NO: 125) and a 3×FLAG tag (SEQ ID NO: 126) at the N-terminus, and a nucleoplasmin NLS sequence (SEQ ID NO: 127) at the C-terminus. Two copies of the NLS sequence were used and operably fused in tandem. Each expression cassette was placed under the control of a cytomegalovirus (CMV) promoter (SEQ ID NO: 306). It is known in the art that a CMV transcriptional enhancer (SEQ ID NO: 307) can also be included in constructs containing a CMV promoter. Guide RNA expression constructs were made, each encoding a single gRNA under the control of a human RNA polymerase III U6 promoter (SEQ ID NO: 308), and introduced into an expression vector. As shown in Table 7, the target regions of selected genes, including RelA, AurkB, GAPDH, LINC01509, HBB, CFTR, HPRT1, TRA, EMX1, and VEGFA, are induced. For the RNA-guided nuclease APG09106.1, specific residues were mutated to increase the nuclease activity of the protein, specifically, the T849 residue of APG09106 was mutated to arginine (SEQ ID NO: 309). This point mutation increased the editing rate in mammalian cells.

[0235] The above constructs were introduced into mammalian cells. One day before transfection, 1x10 5HEK293T cells (Sigma) were plated in 24-well dishes in Dulbecco's modified Eagle's medium (DMEM) + 10% (vol / vol) fetal bovine serum (Gibco) and 1% penicillin-streptomycin (Gibco). The next day when cells were 50-60% confluent, they were co-transfected with 500 ng of RGN expression plasmid + 500 ng of single gRNA expression plasmid using 1.5 µL of Lipofectamine 3000 (Thermo Scientific) per well according to the manufacturer's instructions. After 48 h of growth, total genomic DNA was harvested using a genomic DNA isolation kit (Machery-Nagel) according to the manufacturer's instructions.

[0236] Total genomic DNA was then analyzed to determine the rate of editing in the target genes. Oligonucleotides were generated for use in PCR amplification and subsequent analysis of the amplified genomic target sites (SEQ ID NOs: 310 and 311). All PCR reactions were performed in 20 μL reactions containing 0.5 μM of each primer using 10 μL of 2X Master Mix Phusion High-Fidelity DNA Polymerase (Thermo Scientific). Large genomic regions containing each target gene were first amplified using PCR#1 primers (SEQ ID NOs: 310 and 311) using the following program: 98°C, 1 min; [98°C, 10 sec; 62°C, 15 sec; 72°C, 5 min]; 72°C, 5 min; 12°C, permanent.

[0237] One microliter of this PCR reaction was then further amplified using primers specific for each guide (PCR#2 primers; SEQ ID NOs: 365-370) using the following program: 98°C, 1 min; [98°C, 10 sec; 67°C, 15 sec; 72°C, 30 sec] for 35 cycles; 72°C, 5 min; 12°C. Primers for PCR#2 contain Nextera Read 1 and Read 2 transposase adapter overhang sequences for Illumina sequencing.

[0238] After the second PCR amplification, DNA was cleaned using a PCR cleanup kit (Zymo) according to the manufacturer's instructions and eluted in water. 200-500 ng of purified PCR#2 product was combined with 2 μL of 10X NEB buffer 2 and water in a 20 μL reaction and annealed to form heteroduplex DNA using the following program: 95 °C, 5 min; 95-85 °C, cool at a rate of 2 °C / sec; 85-25 °C, cool at a rate of 0.1 °C / sec; 12 °C, forever. After annealing, 5 μL of DNA was removed as a no enzyme control, 1 μL of T7 endonuclease I (NEB) was added, and the reactions were incubated at 37 °C for 1 h. After incubation, 5× FlashGel loading dye (Lonza) was added, and 5 μL of each reaction and control was analyzed on a 2.2% agarose FlashGel (Lonza) using gel electrophoresis. After visualization of the gel, the percentage of non-homologous end joining (NHEJ) was determined using the following formula: % NHEJ events = 100x[1-(1-fraction cleavage)(1 / 2)], where (fraction cleaved) is defined as (density of digestion product) / (density of digestion product + undigested parent band).

[0239] For some samples, SURVEYOR® was used to analyze the results after expression in mammalian cells. Cells were incubated at 37°C for 72 hours after transfection before genomic DNA extraction. Genomic DNA was extracted using QuickExtract DNA Extraction Solution (Epicentre) according to the manufacturer's protocol. Genomic regions flanking the RGN target site were PCR amplified and the products were purified using QiaQuick Spin Columns (Qiagen) according to the manufacturer's protocol. A total of 200-500 ng of purified PCR product was mixed with 1 μl of 10× Taq DNA polymerase PCR buffer (enzyme) and ultrapure water to a final volume of 10 μl and subjected to a reannealing step to allow heteroduplex formation: 95°C for 10 min, 95°C to 85°C at -2°C / s, 85°C to 25°C at -0.25°C / s, and hold at 25°C for 1 min.

[0240] After reannealing, products were treated with SURVEYOR® Nuclease and SURVEYOR® Enhancer S (Integrated DNA Technologies) according to the manufacturer's recommended protocol and analyzed on 4-20% Novex TBE polyacrylamide gels (Life Technologies). Gels were stained with SYBR Gold DNA stain (Life Technologies) for 10 min and imaged on a GelDoc gel imaging system (Bio-rad). Quantification was based on relative band intensity. Indel percentage was determined by the formula, 100 × (1-(b+c) / (a+b+c)))1 / 2, where a is the integrated intensity of the undigested PCR product and b and c are the integrated intensities of each cleavage product.

[0241] Additionally, products from PCR#2 containing Illumina overhang sequences were subjected to library preparation according to the Illumina 16S Metagenomic Sequencing Library protocol. Deep sequencing was performed on an Illumina Mi-Seq platform by the service provider (MOGene). Typically, 200,000 of 250 bp paired-end reads (2 × 100,000 reads) are generated per amplicon. Reads were analyzed using CRISPResso (Pinello, et al. 2016 Nature Biotech, 34:695-697) to calculate editing rates. Output alignments were hand-cured to confirm insertion and deletion sites as well as identify microhomology sites at the recombination sites. The overall editing, deletion, and insertion rates for each sample are shown in Table 7. All experiments were performed in human cells. "Target" is the target sequence within the gene target. For each target sequence, the guide RNA contained a complementary RNA spacer sequence and the appropriate sgRNA depending on the RGN used. Selected decompositions of the experiments with guide RNA are shown in Tables 8.1 and 8.2.

[0242] [Table 7-1]

[0243] [Table 7-2]

[0244] Tables 8.1 and 8.2 show the specific insertions and deletions for each guide. In these tables, target sequences are identified in bold capital letters. The 8mer PAM region is double underlined and the primary recognition nucleotides are shown in bold. Insertions are identified in lower case letters. Deletions are indicated with dashes (---). INDEL positions are calculated from the PAM-proximal end of the target sequence, with the end being position 0. Positions on the target side of the edge are positive (+) and positions on the PAM side of the edge are negative (-).

[0245] [Table 8]

[0246] [Table 9]

[0247] Example 6: Demonstration of gene editing activity in plant cells The RNA-guided nuclease activity of the RGN of the present invention is demonstrated in plant cells using a protocol modified from Li, et al., 2013 (Nat. Biotech. 31:688-691). Briefly, the plant codon-optimized version of the RGN of the present invention (SEQ ID NO: 1, 9, 16, 23, 30, 38, 46, 54, 61, 69, 75, 82, 89, 95, 103, 110, or 117) is operably linked to a nucleic acid sequence encoding an N-terminal SV40 nuclear localization signal and cloned behind a strong constitutive 35S promoter in a transient transformation vector. sgRNAs targeting one or more sites of a plant PDS gene adjacent to an appropriate PAM sequence are cloned behind a plant U6 promoter in a second transient expression vector. The expression vector is introduced into Nicotiana benthamiana mesophyll protoplasts using PEG-mediated transformation. Transformed protoplasts are incubated in the dark for up to 36 h. Genomic DNA was isolated from protoplasts using the DNeasy Plant Mini Kit (Qiagen). Genomic regions flanking the RGN target site are PCR amplified and the products are purified using QiaQuick Spin Columns (Qiagen) according to the manufacturer's protocol. A total of 200–500 ng of purified PCR product is mixed with 1 μl of 10× Taq DNA polymerase PCR buffer (enzyme) and ultrapure water to a final volume of 10 μl and subjected to a reannealing step to allow heteroduplex formation: 95 °C for 10 min, 95 °C to 85 °C, ramp at -2 °C / s, 85 °C to 25 °C, ramp at -0.25 °C / s, and hold at 25 °C for 1 min.

[0248] After reannealing, the products were treated with SURVEYOR Nuclease and SURVEYOR Enhancer S (Integrated DNA Technologies) according to the manufacturer's recommended protocol, and then gels were stained with SYBR Gold DNA stain (Life Technologies) for 10 min on 4-20% Novex TBE polyacrylamide gels and imaged on a GelDoc gel imaging system (Bio-rad). Quantification was based on relative band intensity. Indel percentages were determined by the formula: 100 × (1-(1-(b+c) / (a+b+c))1 / 2), where a is the integrated intensity of the undigested PCR product and b and c are the integrated intensities of each cleavage product.

[0249] Example 7: Disease Target Identification A database of clinical variants was obtained from the NCBI ClinVar database. The database is available worldwide at the NCBI ClinVar website. Pathogenic single nucleotide polymorphisms (SNPs) were identified from this list. Genomic locus information was used to identify CRISPR targets in the overlapping and surrounding regions of each SNP. A selection of SNPs that can be corrected using base editing in combination with the RGN system including APG09748 or APG09106.1 to target the causative mutation ("Casl Mut.") is shown in Table 9.1. A selection of SNPs that can be corrected using base editing in combination with the RGN system including APG06646.1 or APG01658.1 to target the causative mutation ("Casl Mut.") is shown in Table 9.2. In both Tables 9.1 and 9.2, only one alias for each disease is listed. "RS#" corresponds to the RS accession number via the SNP database on the NCBI website. The chromosome accession numbers provide accession reference information found from the NCBI website. Tables 9.1 and 9.2 also provide, for each disease, genomic target sequence information suitable for the RGN system APG09748 or APG09106.1 (Table 9.1), or APG06646.1 or APG01658.1 (Table 9.2). The target sequence information also provides protospacer sequences for the production of sgRNAs required for the corresponding RGNs of the present invention.

[0250] [Table 10-1]

[0251] [Table 10-2]

[0252] [Table 10-3]

[0253] [Table 11-1]

[0254]

Table 11-2

[0255]

Table 11-3

[0256]

Table 11-4

[0257]

Table 11-5

[0258]

Table 11-6

[0259]

Table 11-7

[0260] Example 8: Targeted mutations causing Friedreich ataxia The expansion of the triple repeat sequence that causes Friedreich ataxia (FRDA) occurs at a specific locus in the FXN gene, called the FRDA instability region. RNA-guided nucleases (RGNs) can be used to excise the unstable region in FRDA patient cells. This approach requires 1) an RGN and a guide RNA sequence that can be programmed to target an allele in the human genome; and 2) a delivery approach for the RGN and guide sequence. Many nucleases used for genome editing, such as the commonly used Cas9 nuclease (SpCas9) from S. pyogenes, are too large to be packaged into an adeno-associated virus (AAV) vector. This makes a viable approach using SpCas9 unlikely.

[0261] The compact RNA-guided nucleases of the present invention, such as APG09748, APG09106.1, and APG06646.1, are specifically suitable for excision of FRDA unstable regions. Each RGN has a PAM requirement that is in the vicinity of the FRDA unstable region. Furthermore, each of these RGNs can be packaged into an AAV vector together with a guide RNA. Although a second vector would be required to pack two guide RNAs, this approach is advantageous compared to that required for larger nucleases such as SpCas9. SpCas9 requires the protein sequence to be split between two vectors.

[0262] Table 10 shows the location of the genomic target sequence suitable for targeting APG09748, APG09106.1, or APG06646.1 to the 5' and 3' flanks of the FRDA unstable region, as well as the sequence of the sgRNA for the genomic target. Once inside the locus, the RGN excises the FA unstable region. Excision of the region can be confirmed by Illumina sequencing of the locus.

[0263] [Table 12]

[0264] Example 9: Targeting the Mutation Causing Sickle Cell Disease Target sequences within the BCL11A enhancer region (SEQ ID NO: 220) may provide a mechanism for increasing fetal hemoglobin (HbF) to cure or alleviate the symptoms of sickle cell disease. For example, genome-wide association studies have identified a series of genetic mutations in BCL11A that are associated with elevated HbF levels. These mutations are a collection of SNPs found in non-coding regions of BCL11A that function as stage-specific lineage-restricted enhancer regions. Further studies have revealed that this BCL11A enhancer is required in erythroid cells for BCL11A expression (Bauer et al, (2013) Science 343:253-257, incorporated herein by reference). The enhancer region is found within intron 2 of the BCL11A gene, and three regions of DNaseI hypersensitivity in intron 2 (indicating chromatin states often associated with regulatable potential) have been identified. These three regions were identified as "+62", "+58" and "+55" according to their distance in kilobases from the transcription start site of BCL11A. These enhancer regions are approximately 350 (+55); 550 (+58); and 350 (+62) nucleotides in length (Bauer et al., 2013).

[0265] Example 9.1: Identifying a Preferred RGN System Here, we describe a potential treatment for β-hemoglobinopathies using the RGN system to disrupt the binding of BCL11A to its binding site within the HBB locus, a gene involved in β-globin production in adult hemoglobin. This approach uses NHEJ, which is more efficient in mammalian cells. Furthermore, this approach uses a nuclease of a small enough size that it can be packaged into a single AAV vector for in vivo delivery.

[0266] The GATA1 enhancer motif in the human BCL11A enhancer region (SEQ ID NO: 220) is an ideal target for disruption using RNA-guided nucleases (RGNs) to reduce BCL11A expression with concomitant re-expression of HbF in adult human erythrocytes (Wu et al. (2019) Nat Med 387:2554). Several PAM sequences compatible with APG09748 or APG09106.1 are readily apparent in the locus surrounding this GATA1 site. These nucleases have a PAM sequence of 5'-DTTN-3' (SEQ ID NO: 60) and are compact in size, allowing their delivery with the appropriate guide RNA in a single AAV or adenoviral vector. In addition to its size, APG06646.1 has minimal PAM requirements (SEQ ID NO: 109) making it suitable for this approach. This delivery approach offers multiple advantages compared to others, such as access to hematopoietic stem cells and a well-established safety profile and manufacturing technology.

[0267] The commonly used Cas9 nuclease from S.pyogenes (SpyCas9) requires a PAM sequence of 5'-NGG-3' (SEQ ID NO: 101), some of which are located near the GATA1 motif. However, the size of SpyCas9 precludes packaging into a single AAV or adenovirus vector, thus forgoing the above-mentioned advantages of this approach. Dual delivery strategies can be employed, but they add significant manufacturing complexity and cost. Furthermore, dual viral vector delivery significantly reduces the efficiency of gene correction, since it requires infection by both vectors for successful editing in a given cell.

[0268] Expression cassettes encoding human codon-optimized APG09748 (SEQ ID NO: 409), APG09106.1 (SEQ ID NO: 410), or APG06646.1 (SEQ ID NO: 415) are produced similarly to that described in Example 5. Expression cassettes expressing guide RNAs for RNAGNAPG09748, APG09106.1, or APG06646.1 are also produced. These guide RNAs contain 1) a protospacer sequence complementary to either the non-coding or coding DNA strand within the BCL11A enhancer locus (target sequence), and 2) an RNA sequence required for the association of the guide RNA with the RGN. Several potential PAM sequences for targeting by each RGN surround the BCL11A GATA1 enhancer motif, so several potential guide RNA constructs are generated to determine the best protospacer sequence that results in strong cleavage and disruption of the BCL11A GATA1 enhancer sequence via NHEJ. The target genomic sequences in Table 11 are evaluated using the sgRNAs shown in Table 11.

[0269] [Table 13]

[0270] To evaluate the efficiency of APG09748, APG09106.1, or APG06646.1 to generate insertions or deletions that disrupt the BCL11A enhancer region, a human cell line such as human embryonic kidney cells (HEK cells) is used. A DNA vector containing an RGN expression cassette (e.g., as described in Example 5) is generated. A separate vector is also generated that contains an expression cassette that includes the coding sequence of a guide RNA sequence in Table 11. Such an expression cassette can further include a human RNA polymerase III U6 promoter (SEQ ID NO: 308), as described in Example 5. Alternatively, a single vector can be used that contains both RGN and guide RNA expression cassettes. The vector is introduced into HEK cells using standard techniques, such as those described in Example 5, and the cells are cultured for 1-3 days. After this culture period, genomic DNA is isolated and the frequency of insertions or deletions is determined using T7 endonuclease I digestion and / or direct DNA sequencing, as described in Example 5.

[0271] The DNA region containing the target BCL11A region is amplified by PCR using primers containing Illumina Nextera XT overhang sequences. These PCR amplicons are either examined for NHEJ formation using T7 endonuclease I digestion or undergo library preparation following the Illumina 16S Metagenomic Sequencing Library protocol or similar next generation sequencing (NGS) library preparation. Following deep sequencing, the generated reads are analyzed by CRISPRESSO to calculate the rate of editing. The output alignments are manually curated to confirm insertion and deletion sites. This analysis identifies the preferred RGNs and the corresponding preferred guide RNAs (sgRNAs). As a result of this analysis, either APG09748, APG09106.1, or APG06646.1 are equally preferred, or one RGN is deemed the most preferred. Furthermore, the analysis may determine that there are multiple preferred guide RNAs, or that all target genomic sequences in Table 17 are equally preferred.

[0272] Example 9.2: Fetal hemoglobin expression study In this example, APG09748, APG09106.1, or APG06646.1, which contain insertions or deletions that disrupt the BCL11A enhancer region, are assayed for fetal hemoglobin expression. + Hematopoietic stem cells (HSCs) are used. These HSCs are cultured and a vector containing an expression cassette containing the coding region of the preferred RGN and the preferred sgRNA is introduced using a method similar to that described in Example 5. Alternatively, electroporation may be used. After electroporation, these cells are differentiated in vitro into erythrocytes using established protocols (e.g., Giarratana et al. (2004) Nat Biotechnology 23:69-74, incorporated herein by reference). Expression of HbF is then measured using Western blotting with anti-human HbF antibody or quantified via high performance liquid chromatography (HPLC). Successful disruption of the BCL11A enhancer locus is expected to increase HbF production compared to HSCs electroporated with RGN alone, but without guidance.

[0273] Example 9.3: Sickle Cell Reduction Study In this experiment, APG09748, APG09106.1, or APG06646.1, which have insertions or deletions that disrupt the BCL11A enhancer region, are assayed for reduction in sickle cell formation. +Hematopoietic stem cells (HSCs) are used. These HSCs are cultured and transfected with a vector containing an expression cassette containing the coding region of the preferred RGN and the preferred sgRNA using a method similar to that described in Example 5. Alternatively, electroporation may be used. After electroporation, these cells are differentiated in vitro into erythrocytes using established protocols (Giarratana et al. (2004) Nat Biotechnology 23:69-74). Expression of HbF is then measured using Western blotting with anti-human HbF antibodies or quantified via high performance liquid chromatography (HPLC). Successful disruption of the BCL11A enhancer locus is expected to increase HbF production compared to HSCs electroporated with RGN alone, but without guidance.

[0274] Sickle cell formation is induced by adding metabisulfite to these differentiated red blood cells. The number of sickle and normal red blood cells is counted under a microscope. The number of sickle cells is expected to be lower in cells treated with APG09748, APG09106.1, or APG06646.1 + sgRNA than in cells untreated or treated with RGN alone.

[0275] Example 9.4: Validation of Disease Treatment in Mouse Models To evaluate the efficacy of disruption of the BCL11A locus by APG09748, APG09106.1, or APG06646.1, a suitable humanized mouse model of sickle cell anemia is used. Expression cassettes encoding the preferred RGN and the preferred sgRNA are packaged into an AAV vector or an adenovirus vector. In particular, the adenovirus type Ad5 / 35 is effective in targeting HSCs. A suitable mouse model containing a humanized HBB locus with sickle cell alleles is selected, such as B6;FVB-Tg(LCR-HBA2,LCR-HBB*E26K)53Hhb / J or B6.Cg-Hbatm1Paz btm1Tow Tg(HBA-HBBs)41Paz / HhbJ. These mice are treated with granulocyte colony-stimulating factor alone or in combination with plerixar for mobilization of HSCs into the blood. AAV or adenovirus carrying RGN and guide plasmids are then injected intravenously and the mice are allowed to recover for one week. Blood obtained from these mice is tested in an in vitro sickling assay using metabisulfite, and the mice are followed longitudinally to monitor mortality and hematopoietic function. Treatment with AAV or adenovirus carrying RGN and guide RNA is expected to ameliorate sickling, mortality, and hematopoietic function compared to mice treated with viruses lacking both expression cassettes or with viruses carrying the RGN expression cassette alone.

[0276] Example 10: Base editing activity in mammalian cells An expression cassette producing a cytidine deaminase-RGN fusion protein was constructed as follows: The coding sequence of RGN (APG06646.1, as described herein, and APG08290.1, described in U.S. Patent Application Publication No. 2019 / 0367949 and International Publication No. WO2019 / 236566, each of which is incorporated herein by reference in its entirety, was codon optimized for mammalian expression and mutated to function as a nickase (SEQ ID NOs: 128 and 262, respectively). This coding sequence was introduced into an expression cassette that generated a fusion protein (SEQ ID NOs: 129-132) that contained an NLS at its N-terminus (SEQ ID NO: 125), operably linked at its C-terminus to a 3xFLAG tag (SEQ ID NO: 126), and operably linked at its C-terminus to a cytidine deaminase. PCT / US2019 / 068079 is operably linked at its C-terminus to an amino acid linker (SEQ ID NO: 133) operably linked at its C-terminus to an RGN nickase and operably linked at its C-terminus to a second NLS (SEQ ID NO: 127), which is incorporated herein by reference in its entirety. These expression cassettes were each introduced into a pTwist CMV vector (Twist Bioscience) capable of driving expression of the fusion protein in mammalian cells. Separate vectors were also generated that express guide RNAs under the control of the human U6 promoter in mammalian cells. These guide RNAs (SEQ ID NOs: 134-136 for nAPG06646.1 and SEQ ID NOs: 263-265 for nAPG08290.1) can guide the deaminase-nRGN fusion protein, or RGN itself, to a target genome sequence for base editing or gene editing, respectively.

[0277] 500 ng of cytidine deaminase-RGN expression plasmid or standard RGN expression plasmid and 500 ng of guide RNA expression plasmid were co-transfected into HEK293FT cells at 75-90% confluence in 24-well plates using Lipofectamine 2000 reagent (Life Technologies). The cells were then incubated at 37 °C for 72 h. Genomic DNA was then extracted using NucleoSpin 96 Tissue (Macherey-Nagel) according to the manufacturer's protocol. Genomic regions flanking the guide RNA target sites were PCR amplified and the products were purified using ZR-96 DNA Clean & Concentrator (Zymo Research) according to the manufacturer's protocol. The purified PCR products were then sent for next-generation sequencing on an Illumina Miq (2 × 250). Results were analyzed for indel formation or specific cytosine mutations.

[0278] Table 12 below shows the actual formation of cytidine base editing and each construct and guide combination. Interestingly, when comparing the activity of RGN APG06646.1 with that of cytidine deaminase-nAPG06646.1 using the same guide RNA, up to 20 times higher cytidine base editing than gene editing was observed. These results demonstrate that RGNs with low nuclease activity at a specific target site can be efficient base editing at that site. Furthermore, since indel formation in base editing applications is often an undesirable outcome, RGNs with low nuclease activity at a site are preferred for base editing applications.

[0279] [Table 14]

[0280] Example 11: Trans ssDNA cleavage 11.1 Determining assay conditions for trans DNA cleavage Purified APG09748 (set forth as SEQ ID NO: 137 and described in International Application No. PCT / US2019 / 068079, incorporated herein by reference in its entirety) was incubated with single guide RNA (sgRNA) in Cutsmart buffer (New England Biolabs B7204S) at a final concentration of 50 nM nuclease and either 100 nM sgRNA or 200 nM nuclease for 10 minutes. These RNP solutions were added to a solution of ssDNA (target or mismatch negative control ssDNA) and reporter probe at a final concentration of 10 nM at a final concentration of 250 nM in 1.5x Cutsmart buffer (New England Biolabs B7204S). The reporter probes (TB0125 and TB0089, shown as SEQ ID NOs: 138 and 139, respectively) contain a fluorescent dye (56-FAM for TB0125 and Cy5 for TB0089) at the 5' end, a quencher (3IABkFQ for TB0125 and 3IAbRQSp for TB0089) at the 3' end, and an optional internal quencher (the internal quencher ZEN is present only on TB0125). Cleavage of the reporter probe results in dequenching of the fluorescent dye and thus an increase in the fluorescent signal. To monitor the fluorescence intensity, 10 μl of each reaction was incubated in a Corning low volume 384-well microplate at 30° C. in a microplate reader (CLARIOSTAR Plus).

[0281] A number of conditions were explored to determine suitable parameters for this assay. To determine whether there was an effect of quenched probe design or fluorophore properties, two such reporters were included as a mixture in each reaction. They were at the same concentration as each other in any given reaction. In all cases, the control or target ssDNA concentration (LE201 or LE205, shown as SEQ ID NOs: 140 and 141, respectively) was 10 nM. RNP names refer to nuclease and target as shown in Table 13 below.

[0282] [Table 15]

[0283] The results are shown in Table 14 below.

[0284] [Table 16-1]

[0285] [Table 16-2]

[0286] From this experiment, it was concluded that a 100 nM concentration of RNP generally results in a higher cleavage rate of the reporter probe than an RNP concentration of 25 nM. In general, reporter cleavage rates are higher at higher concentrations of reporter oligonucleotides up to a reporter concentration of 250-500 nM, with little benefit observed from further increases in reporter concentration. Of note, for the TB0089 reporter (detected in the Cy5 channel), there are substantially high levels of background activity that prevent target differentiation, especially at reporter concentrations above 250 nM. It was therefore concluded that reporter concentrations above 250 nM are not beneficial. Both probes appear to be suitable for observing target recognition by RNPs.

[0287] 11.2 Effect of G09748 trans DNA cleavage and purification on non-specific activity Purified APG09748 was incubated with single guide RNA (sgRNA) in 1× CutSmart buffer (New England Biolabs B7204S) with a final concentration of 200 nM nuclease and 400 nM sgRNA at 37° C. for 10 min, and these RNP solutions were mixed with ssDNA (target or mismatch negative control ssDNA) at a final concentration of 250 nM in 1.5× Cutsmart buffer (New England Biolabs B7204S) at 37° C. The reporter probe contains a fluorescent dye at the 5′ end and a quencher at the 3′ end. Cleavage of the reporter probe results in dequenching of the fluorescent dye and thus an increase in the fluorescent signal. To monitor the fluorescence intensity, 10 μl of each reaction was incubated in a Corning low volume 384-well microplate at 37° C. in a microplate reader (CLARIOSTAR Plus).

[0288] Incubation with the target sequence resulted in a substantial increase in fluorescence intensity as a function of time compared to the negative control. The cleavage rate is summarized as the slope of the linear portion of the fluorescence versus time function, as shown in Table 15.

[0289] [Table 17]

[0290] These data demonstrate discrimination of target sequences from negative controls by RNPs that are clearly detectable using the truncated reporter probe.

[0291] 11.3 PAM measurements using parallel plasmid DNA libraries Oligonucleotides (LE00680 and LE00688, shown as SEQ ID NOs: 266 and 267, respectively) containing the target sequence preceded by a 5 nt degenerate PAM sequence (NNNN) were annealed and cloned into the double-digested pUC19 plasmid using NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs). Since each colony resulting from transformation of this reaction corresponds to a clonal plasmid DNA sequence, preparations of plasmid DNA from cultures derived from a single colony are unique plasmid preparations sampled from the original library. Plasmid preparations were obtained from a sampling of 96 colonies. These preparations were individually subjected to Sanger sequencing to verify their PAM sequences.

[0292] Purified APG09748 was incubated with sgRNA (27sg.2) in 1× CutSmart buffer (New England Biolabs B7204S) at final concentrations of 200 nM nuclease and 400 nM sgRNA for 20 min at room temperature.

[0293] These RNP solutions were added to a final concentration of 100 nM to solutions of plasmid DNA targets at a final concentration of 8.3 nM and the TB0125 and TB0089 reporters (listed as SEQ ID NOs: 138 and 139, respectively) at 250 nM and 50 nM, respectively, in 1.5x Cutsmart buffer (New England Biolabs B7204S). To monitor the fluorescence intensity, 10 μl of each reaction was incubated in a Corning low volume 384-well microplate at 37° C. in a microplate reader (CLARIOSTAR Plus).

[0294] Plasmid concentrations in a few samples were below the target value for concentration normalization or the volume of solution was insufficient to deliver the intended target amount to the reaction well. These samples were not excluded from the analysis and are therefore expected to contribute to error through inconsistencies between duplicates. Samples with plasmids that were determined to have cross contamination (multiple traces seen in the PAM region) as assessed by Sanger sequencing or with plasmids that had target sequence changes removed from the analysis, the results of which are not shown below. The results of the analysis are shown in Table 16 below in descending order by gradient in the FAM channel.

[0295] [Table 18-1]

[0296] [Table 18-2]

[0297] [Table 18-3]

[0298] [Table 18-4]

[0299] The sequence with the highest gradient appears to match the predicted PAM (DTTN as set forth in SEQ ID NO:60) measured by a plasmid curing assay previously described in International Apple. PCT / US2019 / 068079 is incorporated by reference in its entirety. In particular, we see a strong preference for "T" at the two nucleotide positions 5' to the target. Surprisingly, the most active PAM site observed in this assay (ATATG, SEQ ID NO:147) does not exactly match the consensus for DTTN (SEQ ID NO:60), suggesting some level of flexibility in this recognition site.

[0300] 11.4. ssDNA cleavage activity in the presence of PCR-amplified DNA PCR amplified targets were generated from genomic DNA (TRAC and VEGF amplicons shown as SEQ ID NOs:225 and 226, respectively) using appropriate primers. The VEGF target (listed as SEQ ID NO:227) was PCR amplified from HEK293T cell genomic DNA with primers having sequences LE573 and LE578 (listed as SEQ ID NOs:228 and 229, respectively). The TRAC target (listed as SEQ ID NO:230) was PCR amplified with LE257 and LE258 (listed as SEQ ID NOs:231 and 232, respectively).

[0301] RNPs were formed by incubating APG09106.1 nuclease and sgRNA described herein at 0.5 μM and 1 μM, respectively, in 1× NEBuffer 2 (New England Biolabs) and incubated at room temperature for 20 min.

[0302] [Table 19]

[0303] Cleavage reactions were performed in 1.5x NEBuffer 2 using 1.5μM ssDNA oligonucleotide reporter with 5'TEX615 label and 3'Iowa Black FQ quencher and 100nM of each PCR product. Cleavage of the reporter probe results in dequenching of the fluorescent dye and thus an increase in the fluorescent signal. To monitor the fluorescence intensity, 10μl of each reaction was incubated in a Corning low volume 384-well microplate at 37°C in a microplate reader (CLARIOSTAR Plus). The results of the kinetic analysis are shown in Table 18.

[0304] [Table 20]

[0305] These results demonstrate specific activation of trans ssDNA cleavage activity in the presence of PCR-amplified DNA from various sources, with activity dependent on the concentration of PCR-amplified substrate.

[0306] 11.5 Use of ssDNA breaks for diagnosis These nucleases, due to their ability to generate an optically detectable signal in the presence of target DNA sequences, promise utility for implementation into diagnostic devices for the detection of infectious disease pathogens such as bacteria, viruses, or fungi.

[0307] Diagnostic procedures may involve the isolation or amplification of nucleic acids from the sample to be tested. It may also be appropriate to use some samples without performing nucleic acid isolation or purification, since they may be present in the sample in sufficient amounts to be detectable without amplification (such as PCR) or free of substances that interfere with detection or signal generation.

[0308] The RNPs formed as described in other examples can then be exposed to the sample (or the sample treated as described in the previous section) together with a reporter, such as the fluorophore- and quencher-modified single-stranded DNA oligonucleotides used in the previous examples, or other types of single-stranded DNA substrates that generate a visible or otherwise easily detectable signal upon cleavage. If fluorophore-quencher conjugated DNA oligonucleotides are used (as in the previous examples), they can be detected using a fluorimeter, as described in the previous examples. To simplify detection, an end-point assay can be performed instead of the kinetic assay described above, meaning that the assay can be performed for a fixed time against positive and negative controls and read out at the end of this time period.

[0309] These reagents can also be integrated into lateral flow test devices that allow for the detection of a given pathogen or specific nucleic acid sequence (such as a diseased allele in an individual) with very little instrumentation. In this assay, the ssDNA reporter can be conjugated to a number of molecules suitable for antibody or affinity reagent capture, such as fluorescein, biotin, and / or digoxigenin.

Claims

1. A nucleic acid molecule comprising a polynucleotide encoding an RNA-guided nuclease (RGN) polypeptide, wherein the polynucleotide comprises a nucleotide sequence encoding an RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

117.

2. The nucleic acid molecule described in claim 1, wherein the RGN polypeptide is capable of binding to a target DNA sequence of a DNA molecule in an RNA guide sequence-specific manner when bound to a guide RNA (gRNA) capable of hybridizing to the target DNA sequence.

3. A nucleic acid molecule described in claim 1 or 2, wherein a polynucleotide encoding an RGN polypeptide is operably linked to a promoter heterologous to the polynucleotide.

4. A nucleic acid molecule described in any one of claims 1 to 3, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:

117.

5. A nucleic acid molecule described in any one of claims 1 to 4, wherein the RGN polypeptide comprises the amino acid sequence of SEQ ID NO:

117.

6. The nucleic acid molecule of any one of claims 1 to 4, wherein the RGN polypeptide is nuclease inactive or capable of functioning as a nickase.

7. The nucleic acid molecule of any one of claims 1 to 6, wherein the RGN polypeptide is functionally fused to a base-editing polypeptide.

8. A vector comprising a nucleic acid molecule as defined in any one of claims 1 to 7.

9. 9. The vector of claim 8, further comprising at least one nucleotide sequence encoding a gRNA capable of hybridizing to a target DNA sequence, wherein the guide RNA comprises a CRISPR RNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO:

118.

10. 10. The vector of claim 9, wherein the gRNA comprises a tracrRNA having at least 95% sequence identity to SEQ ID NO:

119.

11. A cell comprising a nucleic acid molecule as defined in any one of claims 1 to 7, or a vector as defined in any one of claims 8 to 10.

12. 1. A system for binding a target DNA sequence of a DNA molecule, the system comprising: a) one or more polynucleotides comprising a nucleotide sequence encoding one or more guide RNAs (gRNAs) or one or more guide RNAs capable of hybridizing to said target DNA sequence; and b) an RNA-guided nuclease (RGN) polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 117; or a polynucleotide comprising a nucleotide sequence encoding said RGN polypeptide; Including, The above system, wherein one or more guide RNAs are capable of forming a complex with the RGN polypeptide to bind the RGN polypeptide to the target DNA sequence of a DNA molecule.

13. The system described in claim 12, wherein at least one of the nucleotide sequences encoding one or more guide RNAs and encoding an RGN polypeptide is operably linked to a promoter heterologous to the nucleotide sequence.

14. The system of claim 12 or 13, wherein the target DNA sequence is within a cell.

15. A system described in any one of claims 12 to 14, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to sequence number 117.

16. A system described in any one of claims 12 to 15, wherein the RGN polypeptide has the amino acid sequence of SEQ ID NO:

117.

17. The system of any one of claims 12 to 15, wherein the RGN polypeptide is nuclease inactive or capable of functioning as a nickase.

18. A system described in any one of claims 12 to 17, wherein the RGN polypeptide is functionally linked to a base-editing polypeptide.

19. The system of any one of claims 12 to 16, further comprising one or more donor polynucleotides or one or more nucleotide sequences encoding the one or more donor polynucleotides.

20. A system according to any one of claims 12 to 19 for use in binding a target DNA sequence to a DNA molecule.

21. A system described in any one of claims 12 to 19 for use in cleaving or modifying a target DNA sequence of a DNA molecule.

22. 1. A composition for use in cleaving and / or modifying a target DNA sequence of a DNA molecule, comprising: a) an RNA-guided nuclease (RGN) polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 117; and b) one or more guide RNAs capable of targeting the RGN of (a) to a target DNA sequence; Including, The above composition, wherein one or more guide RNAs hybridize to a target DNA sequence, thereby allowing the RGN polypeptide to bind to the target DNA sequence, resulting in cleavage and / or modification of the target DNA sequence.

23. The composition described in claim 22, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to sequence number 117.

24. The composition described in claim 22 or 23, wherein the RGN polypeptide has the amino acid sequence of SEQ ID NO:

117.

25. A composition for use according to any one of claims 22 to 23, wherein the RGN polypeptide is nuclease inactive or functions as a nickase.

26. A composition for use according to any one of claims 22 to 25, wherein the RGN polypeptide is functionally linked to a base-editing polypeptide.

27. A composition for use according to any one of claims 22 to 26, wherein the target DNA sequence is intracellular.

28. 28. The composition for use of claim 27, further comprising the steps of: culturing the cells under conditions in which the RGN polypeptide is expressed and cleaves the target DNA sequence to produce a DNA molecule comprising a modified DNA sequence; and selecting cells comprising the modified target DNA sequence.

29. An RNA-guided nuclease (RGN) polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

117.

30. The RGN polypeptide described in claim 29, wherein the RGN polypeptide has an amino acid sequence having at least 95% sequence identity to SEQ ID NO:

117.

31. The RGN polypeptide described in claim 29 or 30, wherein the RGN polypeptide has the amino acid sequence of SEQ ID NO:

117.

32. The RGN polypeptide described in claim 29 or 30, wherein the RGN polypeptide is nuclease inactive or functions as a nickase.

33. An RGN polypeptide described in any one of claims 29 to 32, wherein the RGN polypeptide is functionally linked to a base-editing polypeptide.

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