CRISPR-cas effector polypeptides and methods of use thereof

Abstract

The present disclosure provides RNA-guided CRISPR-Cas effector proteins, nucleic acids encoding the effector proteins, and compositions comprising the effector proteins. The present disclosure provides ribonucleoprotein complexes comprising: an RNA-guided CRISPR-Cas effector protein of the present disclosure; and a guide RNA. The present disclosure provides methods of modifying a target nucleic acid using an RNA-guided CRISPR-Cas effector protein and a guide RNA of the present disclosure. The present disclosure provides methods of modulating transcription of a target nucleic acid.

Description

[0001] This application is a divisional application of the same-named invention patent application 202080012951.4, filed March 5, 2020.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 815,173, filed March 7, 2019, U.S. Provisional Patent Application No. 62 / 855,739, filed May 31, 2019, U.S. Provisional Patent Application No. 62 / 907,422, filed September 27, 2019, and U.S. Provisional Patent Application No. 62 / 948,470, filed December 16, 2019, each of which is incorporated herein by reference in its entirety.

[0004] INTRODUCTION

[0005] CRISPR-Cas systems include Cas proteins that are involved in acquisition, targeting, and cleavage of foreign DNA or RNA, and guide RNAs that include a segment that binds to a Cas protein and a segment that binds to a target nucleic acid. For example, Class 2 CRISPR-Cas systems contain a single Cas protein that binds to a guide RNA, where the Cas protein binds to and cleaves a targeted nucleic acid. The programmable nature of these systems has facilitated their use as a general technology for modifying target nucleic acids. SUMMARY

[0006] The present disclosure provides RNA-guided CRISPR-Cas effector proteins, nucleic acids encoding the effector proteins, and compositions comprising the effector proteins. The present disclosure provides ribonucleoprotein complexes comprising: an RNA-guided CRISPR-Cas effector protein of the present disclosure; and a guide RNA. The present disclosure provides methods of modifying a target nucleic acid using an RNA-guided CRISPR-Cas effector protein and a guide RNA of the present disclosure. The present disclosure provides methods of modulating transcription of a target nucleic acid. BRIEF DESCRIPTION OF DRAWINGS

[0007] FIG. 1A Size distributions from complete phage genomes of the present study, Lak phage recently reported from a subset of the same sample, and reference sources (all dsDNA genomes from RefSeq v92 and non-artificial assemblies >200kb (Paez-Espino et al. (2016) Nature 536:425).

[0008] FIG. 1BHistogram showing the genomic size distribution of phages with genomes >200 kb from the present study, Lak, and reference genomes. Boxplot and whisker plot of tRNA count per genome as a function of genome size.

[0009] FIG. 2 Phylogenetic tree constructed using terminal endonuclease sequences from the large phage genomes of the present study and related database sequences. The colored regions of the tree indicate large clades of phages, all of which have large genomes.

[0010] FIG. 3 A model is shown that illustrates how the phage-encoded abilities can function to redirect the host’s translation system to produce phage proteins. No large phage can have all of these genes, but many phages have tRNAs (clover shape) and tRNA synthetases (aaRS). Phage proteins with up to 6 ribosomal protein SI domains appear in some genomes. The SI binds mRNA, bringing it into a site on the ribosome where it is decoded. Ribosomal protein S21 (S21) can selectively initiate translation of phage mRNA, and many sequences have N-terminal extensions that can participate in binding RNA (dashed line in ribosome intercalator, based on PDB code 6bu8 and pmid: 29247757 for ribosome and SI structure models). Some phages have initiation factors (IF) and elongation factor G (EF G), and some have rpL7 / L12, which can mediate efficient ribosome binding. Abbreviations: RNA pol, RNA polymerase.

[0011] FIG. 4A Bacteriophage-bacterium interactions involved in CRISPR targeting (cell diagram) are shown.

[0012] FIG. 4B Interaction network showing CRISPR spacers targeted by bacteria (from top to bottom: SEQ ID NOs: 163-164) and encoded by phages (from top to bottom: SEQ ID NOs: 163-164) are shown.

[0013] FIG. 5 Ecosystems with phages and some plasmids with >200 kbp genomes, grouped by sampling site type are shown. Each box represents a phage genome, and boxes are arranged in order of decreasing genome size; size ranges for each site type are listed on the right. Colors indicate putative host phyla based on genome phylogenetic profiles, confirmed by CRISPR targeting (X) or information system gene phylogenetic analysis (T).

[0014] FIG. 6A-6RThe amino acid sequences of examples of Cas12J polypeptides are provided.

[0015] FIG. 7 Nucleotide sequences of the constant region portion of Cas12J guide RNAs are provided (depicted as DNA encoding the RNA). Sequences in bold are the orientation used and / or inferred from working examples (see, e.g., crRNA ‘use sequences’ in Example 3). Sequences separated by “or” are reverse complements of each other.

[0016] FIG. 8 Consensus sequences for Cas12J guide RNAs are depicted.

[0017] FIG. 9 Positions of amino acids in the RuvC-I, RuvC-II, and RuvC-III domains of Cas12J polypeptides are provided that, when substituted, result in Cas12J polypeptides that bind but do not cleave target nucleic acids in the presence of Cas12J guide RNAs.

[0018] FIG. 10 Trees showing various CRISPR-Cas effector protein families are provided.

[0019] FIG. 11A-11C Efficiencies of transformation plasmid interference assays are shown.

[0020] FIG. 12A-12B Proof that Cas12J (e.g., Cas12J-1947455, Cas12J-2071242, and Cas12J-3339380) can cleave linear dsDNA fragments directed by crRNA spacer sequences is shown.

[0021] FIG. 13 Results demonstrating elucidation of PAM sequences are shown.

[0022] FIG. 14A-14C Results positioning RNA sequences to Cas12J CRISPR loci from pBAS::Cas12J-1947455, pBAS::Cas12J-2071242, and pBAS::Cas12J-3339380 are illustrated.

[0023] FIG. 15 Cas12j-2 and Cas12j-3 mediated gene editing in human cells is depicted.

[0024] FIG. 16A-16B Maps of pCas12J-3-hs(FIG. 16A) and pCas12J-2-hs(FIG. 16B) constructs are provided. FIG. 16A ) and pCas12J-2-hs(FIG. 16B) constructs are provided.

[0025] FIGS. 17A-17G present Table 1, which provides nucleotide sequences for pCasl2J-2-hs and pCasl2J-3-hs constructs (from top to bottom: SEQ ID NOs: 161-162).

[0026] FIG. 18 Depiction of trans cleavage of ssDNA by Casl2J activated by binding to DNA.

[0027] FIG. 19A-19F Description of data showing that Casl2J (CasF) is a true CRISPR-Cas system.

[0028] FIG. 20 Presentation of maximum likelihood phylogenetic tree of V subtypes a-k.

[0029] FIG. 21A-21B Presentation of crRNA repeat sequence similarity between various Casl2J crRNAs FIG. 21A ) and Casl2J amino acid sequence identity between various Casl2J proteins FIG. 21B ).

[0030] FIG. 22A-22C Depiction of CasF-3 mediated protection against plasmid transformation.

[0031] FIG. 23A-23D Depiction of CasF cleavage of DNA.

[0032] FIG. 24A-24D Description of purification of apo CasF (CasF protein without guide RNA).

[0033] FIG. 25A-25C Depiction of staggered nicks produced by CasF.

[0034] FIG. 26A-26B Depiction of CasF mediated cleavage of dsDNA and ssDNA.

[0035] FIG. 27A-27B Depiction of results of cleavage assay comparing target strand (TS) and non-target strand (NTS) cleavage efficiency by CasF.

[0036] FIG. 28A-28B Depiction of data showing that CasF trans cleaves ssDNA, not RNA, after cis activation.

[0037] FIG. 29A-29D Depiction of CasF processing of precursor crRNA within RuvC active site.

[0038] FIG. 30A-30C Depiction of CasF-1 and CasF-2 processing of precursor crRNA.

[0039] FIG. 31A-31B Depiction of CasΦ-mediated enhanced green fluorescent protein (EGFP) disruption in HEK293 cells using the following to form a ribonucleoprotein (RNP) complex: a) precursor crRNA

[0040] FIG. 32A-32C Depiction of CasΦ-mediated enhanced green fluorescent protein (EGFP) disruption in HEK293 cells using the following to form a ribonucleoprotein (RNP) complex: a) precursor crRNA

[0041] FIG. 33A-33B Depiction of CasΦ-mediated enhanced green fluorescent protein (EGFP) disruption in HEK293 cells using the following to form a ribonucleoprotein (RNP) complex: a) precursor crRNA

[0042] Figure 34 presents Table 3, which provides a description of some of the plasmids used in Example 7.

[0043] Figure 35 presents Table 4, which provides guide sequences used for the experiments described in Example 7.

[0044] Figure 36 presents Table 5, which provides substrate sequences used for the in vitro experiments described in Example 7.

[0045] Figure 37 presents Table 6, which provides crRNA sequences used for the in vitro experiments described in Example 7.

[0046] DEFINITIONS

[0047] The terms "polynucleotide" and "nucleic acid," used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double- or multi- stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0048] "Hybridizable" or "complementary" or "substantially complementary" means that a nucleic acid (e.g., RNA, DNA) comprises a sequence of nucleotides that enables it to bind, in a sequence-specific, anti-parallel manner (i.e., the nucleic acid specifically binds to a complementary nucleic acid), non-covalently (i.e., forms Watson-Crick base pairs / or G / U base pairs, "anneals" or "hybridizes") to another nucleic acid under appropriate in vitro and / or in vivo conditions of temperature and solution ionic strength. Standard Watson-Crick base pairing includes: adenine (A) pairs with thymidine (T), adenine (A) pairs with uracil (U), and guanine (G) pairs with cytosine (C) [DNA, RNA]. Additionally, with respect to hybridization between two RNA molecules (e.g., dsRNA), and with respect to hybridization of a DNA molecule to an RNA molecule (e.g., when a DNA target nucleic acid base pairs with a guide RNA, etc.): guanine (G) can also pair with uracil (U). For example, G / U base pairing is at least partially responsible for the degeneracy (i.e., redundancy) of the genetic code, e.g., in the case of tRNA anticodon base pairing with codons in mRNA. Thus, in the context of the present disclosure, guanine (G) (e.g., of a dsRNA duplex of a guide RNA molecule; of a guide RNA that base pairs with a target nucleic acid, etc.) is considered complementary to both uracil (U) and adenine (A). For example, when a G / U base pair can form at a given nucleotide position of a dsRNA duplex of a guide RNA molecule, that position is not considered non-complementary, but rather, is considered complementary.

[0049] Hybridization and washing conditions are well known and exemplified in Sambrook, J., Fritsch, E.F., and Maniatis, T. Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989), in particular Chapter 11 and Table 11.1 therein; and Sambrook, J. and Russell, W., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (2001). Conditions of temperature and ionic strength determine the "stringency" of hybridization.

[0050] Hybridization requires that the two nucleic acids contain complementary sequences, although mismatches between bases can occur. Conditions suitable for hybridization between two nucleic acids depend on the length and degree of complementarity of the nucleic acids, which are well-understood variables in the art. The greater the degree of complementarity between two nucleotide sequences, the greater the value of the melting temperature (Tm) of the hybrid of the nucleic acids having those sequences. For hybridization between nucleic acids having short stretches of complementarity (e.g., more than 35 or fewer, 30 or fewer, 25 or fewer, 22 or fewer, 20 or fewer, or 18 or fewer nucleotides of complementarity), the location of the mismatches can become important (see Sambrook et al., supra, 11.7-11.8). Generally, the length of the hybridizable nucleic acid is 8 nucleotides or more (e.g., 10 nucleotides or more, 12 nucleotides or more, 15 nucleotides or more, 20 nucleotides or more, 22 nucleotides or more, 25 nucleotides or more, or 30 nucleotides or more). The temperature, salt concentration of the wash solution, and other conditions can be adjusted as desired depending on factors such as the length and degree of complementarity of the region of complementarity.

[0051] It is understood that the sequence of a polynucleotide need not be 100% complementary to the sequence of its target nucleic acid to be specifically hybridizing or hybridize. Moreover, a polynucleotide can hybridize over one or more segments, such that intervening or adjacent segments are not involved in the hybridization event (e.g., bulges, loop structures, or hairpin structures, etc.). A polynucleotide can comprise 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence complementarity to a target region within the sequence of the target nucleic acid to which it will hybridize. For example, an antisense nucleic acid in which 18 of 20 nucleotides of the antisense compound are complementary to the target region and will therefore specifically hybridize will represent 90 percent complementarity. In this example, the remaining noncomplementary nucleotides can be clustered or interspersed with complementary nucleotides, and need not be contiguous to or located near to complementary nucleotides. Percent complementarity of a particular segment of nucleic acid sequence within a nucleic acid can be determined using any convenient method. Example methods include the BLAST program (Basic Local Alignment Search Tool) and the PowerBLAST program (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656); the Gap program (Wisconsin Sequence Analysis Package, Version 8, for Unix, Genetics Computer Group, University Research Park, Madison Wis.), which uses the Smith and Waterman algorithm (Adv. Appl. Math., 1981, 2, 482-489), for example using default settings, and the like.

[0052] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein and refer to polymeric forms of amino acids of any length (which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids), as well as to polypeptides having modified peptide backbones.

[0053] As used herein, “bind” (e.g., with reference to an RNA binding domain of a polypeptide, to a target nucleic acid, etc.) refers to a non-covalent interaction between macromolecules (e.g., between a protein and a nucleic acid; between a Cas12J polypeptide / guide RNA complex and a target nucleic acid; etc.). When in a non-covalent interaction state, the macromolecules are said to be “associated” or “interacting” or “bound” (e.g., when molecule X is said to be interacting with molecule Y, it means that molecule X is non-covalently bound to molecule Y). Not all components of a binding interaction need be sequence specific (e.g., contact with a phosphate residue in the DNA backbone), but some portions of the binding interaction can be sequence specific. Binding interactions are typically characterized by dissociation constants (Kd) of less than 10 D M, less than 10 -6 M, less than 10 -7 M, less than 10 -8 M, less than 10 -9 M, less than 10 -10 M, less than 10 -11 M, less than 10 -12 M, less than 10 -13 M, less than 10 -14 M, or less than 10 -15 M. “Affinity” refers to the strength of binding, with increased binding affinity correlating to lower Kd. D

[0054] “Binding domain” means a protein domain that is capable of non-covalently binding to another molecule. Binding domains can bind to, for example, DNA molecules (DNA binding domains), RNA molecules (RNA binding domains), and / or protein molecules (protein binding domains). For proteins having protein binding domains, they can in some cases bind to themselves (to form homodimers, homotrimers, etc.) and / or they can bind to one or more regions of one or more different proteins.

[0055] ​The term "conservative amino acid substitution" refers to the interchangeability of amino acid residues in a protein having similar side chains. For example, one group of amino acids having aliphatic side chains consists of glycine, alanine, valine, leucine and isoleucine; a group of amino acids with aliphatic-hydroxyl side chains consists of serine and threonine; a group of amino acids with amide-containing side chains consists of asparagine and glutamine; a group of amino acids with aromatic side chains consists of phenylalanine, tyrosine, and tryptophan; a group of amino acids with basic side chains consists of lysine, arginine, and histidine; a group of amino acids with acidic side chains consists of glutamic acid and aspartic acid; and a group of amino acids with sulfur-containing side chains consists of cysteine and methionine. Exemplary conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine-glycine, and asparagine-glutamine.

[0056] A polynucleotide or polypeptide has a certain percent "sequence identity" to another polynucleotide or polypeptide when compared, the percentage of bases or amino acids that are the same and in the same relative positions when the sequences are aligned. Sequence identity can be determined in a number of different ways. To determine sequence identity, sequences can be aligned using various convenient methods and computer programs (e.g., BLAST, T-COFFEE, MUSCLE, MAFFT, etc.), which are available on the World Wide Web at sites including ncbi.nlm.nih.gov / BLAST, ebi.ac.uk / Tools / msa / tcoffee / , ebi.ac.uk / Tools / msa / muscle / , mafft.cbrc.jp / alignment / software / . See, e.g., Altschul et al. (1990), J. Mol. Bioi. 215:403-10.

[0057] A DNA sequence that "encodes" a particular RNA is a sequence of DNA nucleotides that is transcribed into RNA. A DNA polynucleotide can encode an RNA (mRNA) that is translated into a protein (and thus, both the DNA and the mRNA encode the protein), or a DNA polynucleotide can encode an RNA that is not translated into a protein (e.g., tRNA, rRNA, microRNA (miRNA), "non-coding" RNA (ncRNA), guide RNA, etc.).

[0058] A "protein-encoding sequence" or a sequence that encodes a particular protein or polypeptide is a nucleotide sequence that is transcribed into mRNA (for DNA) and translated into a polypeptide (for mRNA) in vitro or in vivo when placed under the control of appropriate regulatory sequences.

[0059] The terms "DNA regulatory sequence," "control element," and "regulatory element," used interchangeably herein, refer to transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, protein degradation signals, and the like, that provide for and / or regulate the transcription of non-coding sequences (e.g., guide RNAs) or coding sequences (e.g., RNA-guided endonucleases, GeoCas9 polypeptides, GeoCas9 fusion polypeptides, and the like) and / or regulate translation of encoded polypeptides.

[0060] As used herein, a "promoter" or "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase and initiating transcription of a downstream (3' direction) coding or non-coding sequence. For purposes of the present disclosure, the promoter sequence is bound at its 3' end by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements

[0061] As used herein, the term "naturally occurring" or "unmodified" or "wild type" as applied to a nucleic acid, polypeptide, cell, or organism refers to a nucleic acid, polypeptide, cell, or organism found in nature. For example, a polypeptide or polynucleotide sequence present in an organism that can be isolated from a source in nature is naturally occurring.

[0062] As used herein, the term "fusion" as applied to a nucleic acid or polypeptide refers to two components defined by structures derived from different sources. For example, "fusion" is used in the context of a fusion polypeptide (e.g., a fusion Casl2J protein) includes amino acid sequences derived from different polypeptides. A fusion polypeptide can comprise modified or naturally occurring polypeptide sequences (e.g., a first amino acid sequence from a modified or unmodified Casl2J protein; and a second amino acid sequence from a modified or unmodified protein different from a Casl2J protein, etc.). Likewise, "fusion" in the context of a polynucleotide encoding a fusion polypeptide includes nucleotide sequences derived from different coding regions (e.g., a first nucleotide sequence encoding a modified or unmodified Casl2J protein; and a second nucleotide sequence encoding a polypeptide different from a Casl2J protein).

[0063] The term "fusion polypeptide" refers to a polypeptide that is typically made by the combination (i.e., "fusion") of two otherwise separate segments of an amino acid sequence, usually by artificial intervention.

[0064] As used herein, “heterologous” means a nucleotide or polypeptide sequence that is not found in the native nucleic acid or protein, respectively. For example, in some cases, in a variant Casl2J protein of the disclosure, a portion of a naturally occurring Casl2J polypeptide (or a variant thereof) can be fused to a heterologous polypeptide (i.e., an amino acid sequence from a protein that is different from the Casl2J polypeptide or an amino acid sequence from another organism). As another example, a fusion Casl2J polypeptide can comprise all or a portion of a naturally occurring Casl2J polypeptide (or a variant thereof) fused to a heterologous polypeptide (i.e., a polypeptide from a protein that is different from the Casl2J polypeptide or a polypeptide from another organism). The heterologous polypeptide can exhibit an activity (e.g., enzymatic activity) that would also be exhibited by the variant Casl2J protein or fusion Casl2J protein (e.g., biotin ligase activity; nuclear localization; etc.). A heterologous nucleic acid sequence can be linked (e.g., by genetic engineering) to a naturally occurring nucleic acid sequence (or a variant thereof) to produce a nucleotide sequence that encodes a fusion polypeptide (fusion protein).

[0065] As used herein, "recombinant" means that a particular nucleic acid (DNA or RNA) is the product of various combinations of cloning, restriction, polymerase chain reaction (PCR), and / or ligation steps that result in a construct having a structural coding or non-coding sequence that can be distinguished from an endogenous nucleic acid found in a natural system. A DNA sequence encoding a polypeptide can be assembled from cDNA fragments or from a series of synthetic oligonucleotides to provide a synthetic nucleic acid capable of being expressed from a recombinant transcriptional unit contained in a cell or in a cell-free transcription and translation system. Genomic DNA containing the relevant sequences can also be used to form a recombinant gene or transcriptional unit. Sequences of non-translated DNA can be present 5' or 3' to an open reading frame, where such sequences do not interfere with manipulation or expression of the coding region, and can in fact be used to modulate production of a desired product through various mechanisms (see "DNA Regulatory Sequences"). Alternatively, DNA sequences encoding untranslated RNA (e.g., guide RNA) can also be considered recombinant. Thus, for example, the term "recombinant" nucleic acid refers to a nucleic acid that does not occur naturally as a contiguous segment of a sequence, made by the artificial combination of two otherwise separated segments of sequence by human intervention. This artificial combination is often accomplished by means of chemical synthesis, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques. This is typically done to replace a codon with a codon that encodes the same amino acid, a conserved amino acid, or a non-conserved amino acid. Alternatively, this is done to join nucleic acid segments of a desired function together to produce a desired combination of functions. This artificial combination is often accomplished by means of chemical synthesis, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques. When a recombinant polynucleotide encodes a polypeptide, the sequence of the encoded polypeptide can be naturally occurring ("wild type") or can be a variant (e.g., mutant) of a naturally occurring sequence. An example of such a case is DNA encoding a wild type protein, where the DNA sequence is codon optimized for expression of the protein in a cell in which the protein is not naturally found (e.g., a eukaryotic cell) (e.g., expression of a CRISPR / Cas RNA-guided polypeptide, such as Cas12J (e.g., wild type Cas12J; variant Cas12J; fusion Cas12J; etc.) in a eukaryotic cell). Thus, a codon optimized DNA can be recombinant and non-naturally occurring, while the protein encoded by the DNA can have a wild type amino acid sequence.

[0066] Thus, the term "recombinant" polypeptide does not necessarily refer to a polypeptide whose amino acid sequence does not occur in nature. Rather, a "recombinant" polypeptide is encoded by a recombinant non-naturally occurring DNA sequence, but the amino acid sequence of the polypeptide can be naturally occurring ("wild type") or non-naturally occurring (e.g., variant, mutant, etc.). Thus, a "recombinant" polypeptide is the result of human intervention, but can have a naturally occurring amino acid sequence.

[0067] A "vector" or "expression vector" is a replicon, such as a plasmid, phage, virus, artificial chromosome, or cosmid, to which another DNA segment (i.e., "insert") can be attached so as to bring about the replication of the attached segment in a cell.

[0068] An "expression cassette" comprises a DNA coding sequence operably linked to a promoter. "Operably linked" means colocalized in such a way that the components are in a relationship wherein they function in their intended manner. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence (or, conversely, the coding sequence is operably linked to the promoter).

[0069] The terms "recombinant expression vector" or "DNA construct" are used interchangeably herein to refer to a DNA molecule comprising a vector and an insert. Recombinant expression vectors are typically generated for the expression and / or propagation of an insert, or for the construction of other recombinant nucleotide sequences. The insert can or can not be operably linked to a promoter sequence, and can or can not be operably linked to DNA regulatory sequences.

[0070] A cell has been "genetically modified" or "transformed" or "transfected" by the DNA when exogenous DNA or exogenous RNA (e.g., a recombinant expression vector) has been introduced inside the cell. The presence of foreign DNA results in a permanent or temporary genetic change. The transforming DNA can or can not be integrated (covalently linked) to the genome of the cell. For example, in prokaryotic, yeast, and mammalian cells, the transforming DNA can be maintained on an episomal element such as a plasmid. For eukaryotic cells, a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through cell division. The ability of a eukaryotic cell to establish a cell line or clone comprising a population of daughter cells that includes cells containing the transforming DNA demonstrates such stability. A "clone" is a population of cells obtained from a single cell or common ancestor by mitotic division. A "cell line" is a clone of primary cells that is capable of stable growth through many generations in vitro.

[0071] Suitable methods of genetic modification (also called "transformation") include, for example, viral or phage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethylenimine (PEI)-mediated transfection, DEAE-dextran-mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery (see, e.g., Panyam et al. Adv Drug Deliv Rev. 2012 Sep 13. pii: S0169-409X(12)00283-9. doi: 10.1016 / j.addr.2012.09.023), and the like.

[0072] The choice of genetic modification method generally depends on the type of cell to be transformed and the environment in which the transformation is taking place (e.g., in vitro, ex vivo, or in vivo). A general discussion of these methods can be found in Ausubel et al., Short Protocols in Molecular Biology, 3rded., Wiley & Sons, 1995.

[0073] As used herein, a “target nucleic acid” is a polynucleotide (e.g., DNA, such as genomic DNA) that includes a site (“target site” or “target sequence”) targeted by an RNA-guided endonuclease polypeptide (e.g., wild-type Cas12J; variant Cas12J; fusion Cas12J; etc.). The target sequence is the sequence to which a guide sequence of a subject Cas12J guide RNA (e.g., a dual Cas12J guide RNA or a single-molecule Cas12J guide RNA) will hybridize. For example, a target site (or target sequence) 5'-GAGCAUAUC-3' within a target nucleic acid is targeted by (or binds to, hybridizes to, or is complementary to) the sequence 5'-GAUAUGCUC-3'. Suitable hybridization conditions include physiological conditions typically found in a cell. For a double-stranded target nucleic acid, the strand of the target nucleic acid that is complementary to and hybridizes to the guide RNA is referred to as the “complementary strand” or “target strand”; while the strand of the target nucleic acid that is complementary to the “target strand” (and thus not complementary to the guide RNA) is referred to as the “non-target strand” or “non-complementary strand”.

[0074] “Cleavage” means the breakage of the covalent backbone of a target nucleic acid molecule (e.g., RNA, DNA). Cleavage can be initiated by a variety of methods, including but not limited to, enzymatic or chemical hydrolysis of phosphodiester bonds. Single-strand cleavage and double-strand cleavage are both possible, and double-strand cleavage can occur as a result of two distinct single-strand cleavage events.

[0075] “Nuclease” and “endonuclease” are used interchangeably herein to mean an enzyme having catalytic activity for nucleic acid cleavage (e.g., ribonuclease activity (ribose nucleic acid cleavage), deoxyribonuclease activity (deoxyribose nucleic acid cleavage), etc.).

[0076] A “cleavage domain” or “active domain” or “nuclease domain” of a nuclease means the polypeptide sequence or domain within the nuclease that has catalytic activity for nucleic acid cleavage. The cleavage domain can be contained within a single polypeptide chain, or the cleavage activity can result from the association of two (or more) polypeptides. A single nuclease domain can be composed of more than one contiguous amino acid within a given polypeptide.

[0077] The term "stem cell" is used herein to refer to a cell that has the ability to self-renew and to generate differentiated cell types (e.g., plant stem cells, vertebrate stem cells) (see Morrison et al. (1997) Cell 88:287-298). In the context of cellular ontogeny, the adjectives "differentiated" or "differentiating" are relative terms. A "differentiated cell" is a cell that is further down the developmental pathway than the cell being compared. Thus, a pluripotent stem cell (described below) can differentiate into a lineage-restricted progenitor cell (e.g., a mesoderm stem cell), which in turn can differentiate into a further restricted cell (e.g., a neuronal progenitor cell), which can differentiate into a terminally differentiated cell (i.e., a terminally differentiated cell, e.g., a neuron, a cardiomyocyte, etc.), which has a characteristic role in a particular tissue type and which can or can not retain the ability to further proliferate. Stem cells can be characterized by the presence of specific markers (e.g., proteins, RNAs, etc.) and the absence of specific markers. Stem cells can also be identified by in vitro and in vivo functional assays, particularly assays related to the ability of stem cells to generate multiple differentiated progeny.

[0078] Stem cells of interest include pluripotent stem cells (PSCs). The term "pluripotent stem cell" or "PSC" is used herein to mean a stem cell that is capable of generating all cell types of an organism. Thus, a PSC can generate cells of all germ layers of an organism (e.g., endoderm, mesoderm, and ectoderm of a vertebrate). A pluripotent cell is capable of forming a teratoma and contributes to ectoderm, mesoderm, or endoderm tissue in a living organism. A pluripotent stem cell of a plant is capable of generating all cell types of the plant (e.g., cells of roots, stems, leaves, etc.).

[0079] PSCs of animals can be obtained in a variety of different ways. For example, embryonic stem cells (ESCs) are derived from the inner cell mass of an embryo (Thomson et al., Science. 1998 Nov 6;282(5391): 1145-7), while induced pluripotent stem cells (iPSCs) are derived from somatic cells (Takahashi et al., Cell. 2007 Nov 30;131(5):861-72; Takahashi et al., Nat Protoc. 2007;2(12):3081-9; Yu et al., Science. 2007 Dec 21;318(5858): 1917-20. Epub 2007 Nov 20). Because the term PSC refers to pluripotent stem cells regardless of their origin, the term PSC encompasses the terms ESC and iPSC, as well as the term embryonic germ stem cells (EGSCs), which are another example of PSCs. PSCs can be in the form of established cell lines, which can be obtained directly from primary embryonic tissue, or which can be obtained from somatic cells. PSCs can be target cells of the methods described herein.

[0080] "Embryonic stem cells" (ESCs) mean PSCs isolated from an embryo, typically from the inner cell mass of a blastocyst. ESC lines are listed in the NIH Human Embryonic Stem Cell Registry, for example, hES BGN-01, hES BGN-02, hES BGN-03, hES BGN-04 (BresaGen, Inc.); HES-1, HES-2, HES-3, HES-4, HES-5, HES-6 (ES Cell International); Miz-hES1 (MizMedi Hospital-Seoul National University); HSF-1, HSF-6 (University of California at San Francisco); and H1, H7, H9, H13, H14 (Wisconsin Alumni Research Foundation (WiCell Research Institute)). Stem cells of interest also include embryonic stem cells from other primates, such as rhesus monkey stem cells and marmoset stem cells. The stem cells can be obtained from any mammalian species, for example, humans, equines, bovines, porcines, canines, felines, rodents, such as mice, rats, hamsters, primates, and the like (Thomson et al. (1998) Science 282:1145; Thomson et al. (1995) Proc. Natl. Acad. Sci USA 92:7844; Thomson et al. (1996) Biol. Reprod. 55:254; Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998). In culture, ESCs typically grow as flat colonies with large nuclear-cytoplasmic ratios, distinct borders, and prominent nucleoli. In addition, ESCs express SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, and alkaline phosphatase, but not SSEA-1. Examples of methods to generate and characterize ESCs can be found in, for example, U.S. Patent No. 7,029,913, U.S. Patent No. 5,843,780, and U.S. Patent No. 6,200,806, the disclosures of which are incorporated herein by reference. Methods for propagating hESCs in an undifferentiated form are described in WO 99 / 20741, WO 01 / 51616, and WO 03 / 020920.

[0081] "Embryonic germ stem cells" (EGSCs) or "embryonic germ cells" or "EG cells" mean PSCs derived from germ cells and / or germ cell progenitors, such as primordial germ cells (i.e., those that will become sperm and eggs). Embryonic germ cells (EG cells) are believed to have similar properties as embryonic stem cells as described above. Examples of methods to generate and characterize EG cells can be found, for example, in U.S. Patent No. 7,153,684; Matsui, Y. et al. (1992) Cell 70:841; Shamblott, M. et al. (2001) Proc. Natl. Acad. Sci. USA 98:113; Shamblott, M. et al. (1998) Proc. Natl. Acad. Sci. USA, 95:13726; and Koshimizu, U. et al. (1996) Development, 122:1235, the disclosures of which are incorporated herein by reference.

[0082] "Induced pluripotent stem cells" or "iPSCs" mean PSCs derived from non-PSC cells (i.e., cells that are differentiated relative to PSCs). iPSCs can be derived from a variety of different cell types, including terminally differentiated cells. iPSCs have an ES cell-like morphology, growing as flat colonies with large nuclear-cytoplasmic ratios, distinct borders, and prominent nucleoli. In addition, iPSCs express one or more key pluripotency markers known to those of ordinary skill in the art, including, but not limited to, alkaline phosphatase, SSEA3, SSEA4, Soχ2, Oct3 / 4, Nanog, TRA160, TRA181, TDGF 1, Dnmt3b, FoxD3, GDF3, Cyp26al, TERT, and zfp42. Examples of methods to generate and characterize iPSCs can be found, for example, in U.S. Patent Publication Nos. US20090047263, US20090068742, US20090191159, US20090227032, US20090246875, and US20090304646, the disclosures of which are incorporated herein by reference. Typically, to generate iPSCs, a somatic cell is provided with reprogramming factors known in the art (e.g., Oct4, SOχ2, KLF4, MYC, Nanog, Lin28, etc.) to reprogram the somatic cell into a pluripotent stem cell.

[0083] “Somatic cell” means any cell in an organism that would not normally give rise to all types of cells in the organism in the absence of experimental manipulation. In other words, a somatic cell is a cell that has fully differentiated and would not naturally give rise to cells of all three germ layers of the body (i.e., ectoderm, mesoderm, and endoderm). For example, somatic cells would include neurons and neural progenitor cells, where the latter can be able to naturally give rise to all or some cell types of the central nervous system, but not cells of the mesodermal or endodermal lineages.

[0084] “Mitotic cell” means a cell that is undergoing mitosis. Mitosis is the process by which a eukaryotic cell divides the chromosomes in its nucleus into two identical sets in two separate nuclei. This is generally followed by cytokinesis, which divides the nucleus, cytoplasm, organelles, and cell membrane into two cells containing roughly equal shares of these cellular components.

[0085] “Post-mitotic cell” means a cell that has exited from mitosis, i.e., it is “resting,” i.e., it is no longer undergoing division. This resting state can be temporary, i.e., reversible, or it can be permanent.

[0086] “Meiotic cell” means a cell that is undergoing meiosis. Meiosis is the process by which a cell divides its nuclear material in order to produce gametes or spores. Unlike mitosis, in meiosis, chromosomes undergo a recombination step that shuffles genetic material between chromosomes. In addition, the result of meiosis is four (genetically distinct) haploid cells, as compared to two (genetically identical) diploid cells produced by mitosis.

[0087] In some instances, a component (e.g., a nucleic acid component (e.g., a Cas12J guide RNA); a protein component (e.g., a wild-type Cas12J polypeptide; a variant Cas12J polypeptide; a fusion Cas12J polypeptide; etc.); etc.) includes a label moiety. As used herein, the term “label,” “detectable label,” or “label moiety” refers to any moiety that provides for detection of a signal and can vary widely depending on the particular nature of the assay. Label moieties of interest include directly detectable labels (direct labels; e.g., fluorescent labels) and indirectly detectable labels (indirect labels; e.g., binding partner members). Fluorescent labels can be any fluorescent label (e.g., a fluorescent dye (e.g., fluorescein, Texas Red, rhodamine, Labels, etc.), fluorescent proteins (e.g., green fluorescent protein (GFP), enhanced GFP (EGFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), cherry, tomato, orange, and any fluorescent derivative thereof), etc.). Suitable detectable (direct or indirect) labeled portions for the method include any portion detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, chemical, or other means. For example, suitable indirect labels include biotin (a binding pair member) that can be bound by streptavidin (which itself can be directly or indirectly labeled). Labels may also include: radioactive labels (direct labels) (e.g. 3 H, 125 I, 35 S, 14 C or 32 P); enzymes (indirect labeling) (e.g., peroxidase, alkaline phosphatase, galactosidase, luciferase, glucose oxidase, etc.); fluorescent proteins (direct labeling) (e.g., green fluorescent protein, red fluorescent protein, yellow fluorescent protein, and any convenient derivative thereof); metal labels (direct labeling); colorimetric labels; binding pair members; etc. "Partner of a binding pair" or "binding pair member" means one of the first and second parts, wherein the first and second parts have a specific binding affinity to each other. Suitable binding pairs include, but are not limited to: antigen / antibody (e.g., digoxigenin / anti-digoxigenin, dinitrophenyl (DNP) / anti-DNP, dansyl-X-antidansyl, luciferin / anti-luciferin, fluorescein / anti-fluorescein, and rhodamine / anti-rhodamine), biotin / anti-biotin (or biotin / streptavidin), and calmodulin-binding protein (CBP) / calmodulin. Any binding pair member may be suitable as an indirectly detectable labeling moiety.

[0088] Any given component or combination of components may be unlabeled or detectably labeled with a tagged portion. In some cases, when two or more components are labeled, the components may be labeled with tagged portions that are distinguishable from each other.

[0089] General methods in molecular and cellular biochemistry can be found in standard textbooks, such as Molecular Cloning: A Laboratory Manual, 3rded. (Sambrook et al., HaRBor Laboratory Press 2001); Short Protocols in Molecular Biology, 4thed. (Ausubel et al., eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al., eds., Academic Press 1999); Viral Vectors (Kaplift and Loewy, eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits, ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle and Griffiths, John Wiley & Sons 1998), the disclosures of which are incorporated herein by reference.

[0090] As used herein, the terms "treatment" and "treating," and the like, refer to obtaining a desired pharmacologic and / or physiologic effect. The effect can be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or can be therapeutic in terms of a partial or complete cure for a disease and / or adverse effect attributable to the disease. "Treatment," as used herein, covers any treatment of a disease in a mammal, e.g., in a human, and includes: (a) preventing the disease from occurring in an individual which can be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.

[0091] The terms "individual," "subject," "host," and "patient," used interchangeably herein, refer to an individual organism, such as a mammal, including, but not limited to, murines, simians, humans, non-human primates, ungulates, felines, canines, bovines, ovines, mammalian farm animals, mammalian sport animals, and mammalian pets.

[0092] Before further description of the application, it should be understood that the application is not limited to the particular embodiments described, as such can of course vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of the application will be limited only by the appended claims.

[0093] In providing ranges of values, it should be understood that, unless the context clearly dictates otherwise, each intervening value between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the scope of the application. The upper and lower limits of these smaller ranges can independently be included in the smaller ranges and are also encompassed within the application, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the application.

[0094] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present application, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0095] It must be noted that, as used herein and in the appended claims, the singular form "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a Cas12J CRISPR-Cas effector polypeptide" includes a plurality of such polypeptides, and reference to "a guide RNA" includes reference to one or more guide RNAs known to those skilled in the art and equivalents thereof, and the like. It should also be noted that the claims can be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for using such exclusive terminology to delineate the present scope.

[0096] It should be appreciated that certain features of the application, which are, for clarity, described in the context of separate embodiments, can also be provided in combination in a single embodiment. Conversely, various features of the application, which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable subcombination. The application specifically contemplates the various embodiments of the application in all possible technical combinations. The disclosure hereby expressly incorporates the entirety of any other publications cited herein as if each were individually and specifically incorporated by reference in their entirety. Additionally, the application specifically contemplates all subcombinations of the various embodiments and elements thereof as if each such subcombination was individually and specifically herein preemptively asserted.

[0097] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as a admission that the present application is not entitled to antedate such publications by virtue of prior application. Further, the dates of publication provided can be different from the actual publication dates which can need to be independently confirmed. DETAILED DESCRIPTION

[0098] The present disclosure provides RNA-guided CRISPR-Cas effector proteins, referred to herein as "Casl2J" polypeptides, "CasΦ" polypeptides, or "CasXS" polypeptides; nucleic acids encoding the effector proteins; and compositions comprising the effector proteins. The present disclosure provides ribonucleoprotein complexes comprising: a Casl2J polypeptide of the present disclosure; and a guide RNA. The present disclosure provides methods of using Casl2J polypeptides of the present disclosure and guide RNAs to modify target nucleic acids. The present disclosure provides methods of modulating transcription of a target nucleic acid.

[0099] The present disclosure provides guide RNAs that bind to and provide sequence specificity for Casl2J proteins (referred to herein as "Casl2J guide RNAs"); nucleic acids encoding Casl2J guide RNAs; and modified host cells comprising Casl2J guide RNAs and / or nucleic acids encoding the same. The provided Casl2J guide RNAs can be used in a number of applications.

[0100] COMPOSITIONS

[0101] CRISPR / CAS12J PROTEINS AND GUIDE RNAs

[0102] Casl2J CRISPR / Cas effector polypeptides (e.g., Casl2J proteins; also referred to as "CasXS polypeptides" or "CasΦ polypeptides") interact (bind) with corresponding guide RNAs (e.g., Casl2J guide RNAs) to form ribonucleoprotein (RNP) complexes that target specific sites in a target nucleic acid (e.g., a target DNA) via base pairing between the guide RNA and a target sequence within the target nucleic acid molecule. The guide RNA includes a nucleotide sequence (a guide sequence) that is complementary to a sequence of the target nucleic acid (a target site). Thus, a Casl2J protein forms a complex with a Casl2J guide RNA, and the guide RNA provides sequence specificity to the RNP complex via the guide sequence. The Casl2J protein of the complex provides site-specific activity. In other words, a Casl2J protein is directed (e.g., stabilized) to a target site within a target nucleic acid sequence (e.g., a chromosomal sequence or an extrachromosomal sequence, e.g., an episomal sequence, a minicircle nucleic acid, a mitochondrial sequence, a chloroplast sequence, etc.) as a result of its association with a guide RNA.

[0103] In some cases, the Casl2J CRISPR / Cas effector polypeptides of the disclosure cleave double stranded DNA or single stranded DNA, but not single stranded RNA, when complexed with a guide RNA.

[0104] In some cases, the Casl2J CRISPR / Cas effector polypeptides of the disclosure catalyze the processing of a precursor crRNA in a magnesium-dependent manner.

[0105] The disclosure provides compositions comprising a Casl2J polypeptide (and / or a nucleic acid comprising a nucleotide sequence encoding the Casl2J polypeptide) (e.g., where the Casl2J polypeptide can be a naturally occurring protein, a nickase Casl2J protein, a catalytically inactive (“dead” Casl2J; also referred to herein as a “dCasl2J protein”), a fusion Casl2J protein, etc.). The disclosure provides compositions comprising a Casl2J guide RNA (and / or a nucleic acid comprising a nucleotide sequence encoding the Casl2J guide RNA). The disclosure provides compositions comprising (a) a Casl2J polypeptide (and / or a nucleic acid encoding the Casl2J polypeptide) (e.g., where the Casl2J polypeptide can be a naturally occurring protein, a nickase Casl2J protein, a dCasl2J protein, a fusion Casl2J protein, etc.) and (b) a Casl2J guide RNA (and / or a nucleic acid encoding the Casl2J guide RNA). The disclosure provides a nucleic acid / protein complex (RNP complex) comprising: (a) a Casl2J polypeptide of the disclosure (e.g., where the Casl2J polypeptide can be a naturally occurring protein, a nickase Casl2J protein, a dCasl2J protein, a fusion Casl2J protein, etc.); and (b) a Casl2J guide RNA.

[0106]

Casl2J Proteins

[0107] Casl2J polypeptides (this term is used interchangeably with the terms “Casl2J proteins,” “CasΦ polypeptides,” and “CasΦ proteins”) can bind and / or modify (e.g., cleave, nick, methylate, demethylate, etc.) target nucleic acids and / or polypeptides associated with target nucleic acids (e.g., methylation or acetylation of histone tails) (e.g., in some cases, the Casl2J proteins include a fusion partner that has activity, and in some cases, the Casl2J proteins provide nuclease activity). In some cases, the Casl2J proteins are naturally occurring proteins (e.g., naturally occurring in a bacteriophage). In other cases, the Casl2J proteins are not naturally occurring polypeptides (e.g., the Casl2J proteins are variant Casl2J proteins (e.g., catalytically inactive Casl2J proteins, fusion Casl2J proteins, etc.).

[0108] A Casl2J polypeptide (e.g., not fused to any heterologous fusion partner) can have a molecular weight of about 65 kilodaltons (kDa) to about 85 kDa. For example, a Casl2J polypeptide can have a molecular weight of about 65 kDa to about 70 kDa, about 70 kDa to about 75 kDa, or about 75 kDa to about 80 kDa. For example, a Casl2J polypeptide can have a molecular weight of about 70 kDa to about 80 kDa.

[0109] An assay to determine whether a given protein interacts with a Casl2J guide RNA can be any convenient binding assay to test binding between a protein and a nucleic acid. Suitable binding assays (e.g., gel shift assays) will be known to those of ordinary skill in the art (e.g., including assays in which a Casl2J guide RNA and a protein are added to a target nucleic acid). An assay to determine whether a protein is active (e.g., to determine whether the protein has nuclease activity to cleave a target nucleic acid and / or some heterologous activity) can be any convenient assay (e.g., any convenient assay to test nucleic acid cleavage). Suitable assays (e.g., cleavage assays) will be known to those of ordinary skill in the art.

[0110] Naturally occurring Casl2J proteins act as endonucleases that catalyze a double-stranded break at a specific sequence in a targeted double-stranded DNA (dsDNA). Sequence specificity is provided by an associated guide RNA that hybridizes to a target sequence within the target DNA. Naturally occurring Casl2J guide RNAs are crRNAs, where the crRNA includes (i) a guide sequence that hybridizes to a target sequence in the target DNA and (ii) a protein-binding segment that includes a stem-loop (hairpin-dsRNA duplex) that binds to a Casl2J protein.

[0111] In some cases, a C12J polypeptide of the disclosure, when complexed with a Casl2J guide RNA, produces a product nucleic acid comprising a 5' overhang following site-specific cleavage of a target nucleic acid. The 5' overhang can be an 8 to 12 nucleotide (nt) overhang. For example, the 5' overhang can be 8 nt, 9 nt, 10 nt, 11 nt, or 12 nt long.

[0112] In some embodiments, a Casl2J protein of a method and / or composition of the disclosure is (or is derived from) a naturally occurring (wild-type) protein. Examples of naturally occurring Casl2J proteins are depicted in FIG. 6A-6R In some cases, a Casl2J protein (of a composition and / or method of the disclosure) includes a Casl2J protein depicted in FIG. 6 (e.g., FIG. 6A-6RAny Cas12J amino acid sequence in any of the following (either of the following) has 20% or higher sequence identity (e.g., 30% or higher, 40% or higher, 50% or higher, 60% or higher, 70% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity). In some cases, the Cas12J protein (of the compositions and / or methods of the present invention) comprises the amino acid sequence depicted in Figure 6 (e.g., FIG. 6A-6R The amino acid sequence of any one of them.

[0113] In some cases, the sequence identity of the Cas12J protein (of the composition and / or method of the present invention) with the amino acid sequence depicted in FIG. 6 (e.g., any Cas12J amino acid sequence depicted in FIG. 6) is higher than its sequence identity with any of the following: Cas12a protein, Cas12b protein, Cas12c protein, Cas12d protein, Cas12e protein, Cas12g protein, Cas12h protein, and Cas12i protein. In some cases, the Cas12J protein (of the compositions and / or methods of the present invention) comprises an amino acid sequence having a RuvC domain (which includes RuvC-I, RuvC-II, and RuvC-III domains), the RuvC domain having a higher sequence identity with the RuvC domain of the amino acid sequence depicted in FIG. 6 (e.g., the RuvC domain of any Cas12J amino acid sequence depicted in FIG. 6) than with the RuvC domain of any of the following: Cas12a protein, Cas12b protein, Cas12c protein, Cas12d protein, Cas12e protein, Cas12g protein, Cas12h protein, and Cas12i protein.

[0114] In some cases, the Cas12J protein (of the compositions and / or methods of the present invention) includes the protein depicted in Figure 6 (e.g., FIG. 6A-6R The RuvC domain (including RuvC-I, RuvC-II, and RuvC-III domains) of any Cas12J amino acid sequence in any of the following (any of the following) has 20% or higher sequence identity (e.g., 30% or higher, 40% or higher, 50% or higher, 60% or higher, 70% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity). In some cases, the Cas12J protein (of the compositions and / or methods of the present invention) comprises an amino acid sequence that is identical to that depicted in Figure 6 (e.g., FIG. 6A-6RThe RuvC domain (which includes RuvC-I, RuvC-II, and RuvC-III domains) of any Cas12J amino acid sequence in any of SEQ ID NOS: 1-3 has an amino acid sequence of 70% or more sequence identity (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity). In some cases, a Cas12J protein (of a composition and / or method of the application) includes a RuvC domain having an amino acid sequence depicted in FIG. 6 (e.g., FIG. 6A-6R The RuvC domain (which includes RuvC-I, RuvC-II, and RuvC-III domains) of any Cas12J amino acid sequence in any of SEQ ID NOS: 1-3.

[0115] In some cases, a guide RNA that binds a Cas12J polypeptide includes a nucleotide sequence (or in some cases the reverse complement thereof) depicted in any of SEQ ID NOS: 4-6. FIG. 7 In some cases, a guide RNA that binds a Cas12J polypeptide includes a nucleotide sequence (or in some cases the reverse complement thereof) depicted in any of SEQ ID NOS: 4-6. FIG. 7 In some cases, a guide RNA that binds a Cas12J polypeptide includes a nucleotide sequence of 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to any of the sequences (or in some cases the reverse complement thereof) depicted in any of SEQ ID NOS: 4-6. In some cases, the guide RNA comprises a nucleotide sequence (N)nX or the reverse complement thereof, where N is any nucleotide, n is an integer from 15 to 30 (e.g., 15 to 20, 17 to 25, 17 to 22, 18 to 22, 18 to 20, 20 to 25, or 25 to 30), and X is a nucleotide sequence of 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to any of the sequences depicted in any of SEQ ID NOS: 4-6.

[0116] In some cases, a guide RNA that binds a Cas12J polypeptide includes a nucleotide sequence of 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to any of the sequences (or in some cases the reverse complement thereof) depicted in any of SEQ ID NOS: 4-6. In some cases, the guide RNA comprises a nucleotide sequence (N)nX or the reverse complement thereof, where N is any nucleotide, n is an integer from 15 to 30 (e.g., 15 to 20, 17 to 25, 17 to 22, 18 to 22, 18 to 20, 20 to 25, or 25 to 30), and X is a nucleotide sequence of 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to any of the sequences depicted in any of SEQ ID NOS: 4-6. FIG. 7 FIG. 7 In some cases, a guide RNA that binds a Cas12J polypeptide includes a nucleotide sequence of 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to any of the sequences (or in some cases the reverse complement thereof) depicted in any of SEQ ID NOS: 4-6. In some cases, the guide RNA comprises a nucleotide sequence (N)nX or the reverse complement thereof, where N is any nucleotide, n is an integer from 15 to 30 (e.g., 15 to 20, 17 to 25, 17 to 22, 18 to 22, 18 to 20, 20 to 25, or 25 to 30), and X is a nucleotide sequence of 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to any of the sequences depicted in any of SEQ ID NOS: 4-6.

[0117] In some cases, a guide RNA that binds a Cas12J polypeptide includes a nucleotide sequence of 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to any of the sequences (or in some cases the reverse complement thereof) depicted in any of SEQ ID NOS: 4-6. In some cases, the guide RNA comprises a nucleotide sequence (N)nX or the reverse complement thereof, where N is any nucleotide, n is an integer from 15 to 30 (e.g., 15 to 20, 17 to 25, 17 to 22, 18 to 22, 18 to 20, 20 to 25, or 25 to 30), and X is a nucleotide sequence of 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to any of the sequences depicted in any of SEQ ID NOS: 4-6.​FIG. 7 In some cases, a guide RNA that binds a Cas12J polypeptide comprises a nucleotide sequence (or in some cases the reverse complement thereof) of any of the sequences depicted in FIG. 7 In some cases, a guide RNA that binds a Cas12J polypeptide comprises a nucleotide sequence of 85% or greater sequence identity (e.g., 90% or greater, 95% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% sequence identity) to any of the sequences depicted in

[0118] In some cases, a guide RNA that binds a Cas12J polypeptide comprises a nucleotide sequence (or in some cases the reverse complement thereof) of any of the sequences depicted in FIG. 7 In some cases, a guide RNA that binds a Cas12J polypeptide comprises a nucleotide sequence X(N)n, where N is any nucleotide, n is an integer from 15 to 30 (e.g., 15 to 20, 17 to 25, 17 to 22, 18 to 22, 18 to 20, 20 to 25, or 25 to 30), and X is any of the nucleotide sequences depicted in FIG. 7 In some cases, a guide RNA that binds a Cas12J polypeptide comprises a nucleotide sequence (or in some cases the reverse complement thereof) of any of the sequences depicted in

[0119] In some cases, a guide RNA that binds a Cas12J polypeptide comprises a nucleotide sequence of 20% or greater sequence identity (e.g., 30% or greater, 40% or greater, 50% or greater, 60% or greater, 70% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% sequence identity) to any of the sequences depicted in FIG. 7 In some cases, a guide RNA that binds a Cas12J polypeptide comprises a nucleotide sequence X(N)n, where N is any nucleotide, n is an integer from 15 to 30 (e.g., 15 to 20, 17 to 25, 17 to 22, 18 to 22, 18 to 20, 20 to 25, or 25 to 30), and X is any of the nucleotide sequences depicted in FIG. 6A-6R In some cases, a guide RNA that binds a Cas12J polypeptide comprises a nucleotide sequence of 20% or greater sequence identity (e.g., 30% or greater, 40% or greater, 50% or greater, 60% or greater, 70% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% sequence identity) to any of the sequences depicted in

[0120] Examples of Cas12J proteins are depicted in FIG. 9 As noted above, Cas12J polypeptides are also referred to herein as “CasΦ polypeptides.” For example:

[0121] (1) designated “Cas12J_1947455” (or FIG. 6A “Cas12J_1947455_11”) in Table 1 and depicted in FIG. 1A; FIG. 6B The Cas12J polypeptide in Table 1 (or “Cas12J_1947455_11”) is also referred to herein as “CasΦ-1”;

[0122] (2) designated “Cas12J_2071242” and depicted in FIG. 1B; FIG. 9 The Cas12J polypeptide in Table 1 (or “Cas12J_2071242”) is also referred to herein as “CasΦ-2”

[0123] (3) designated “Cas12J_3339380” (or FIG. 6D “Cas12J_3339380_12”) in Table 1 and depicted in FIG. 1C; FIG. 6Q The Cas12J polypeptide in Table 1 (or “Cas12J_3339380_12”) is also referred to herein as “CasΦ-3”;

[0124] (4) designated “Cas12J_3877103_16” and depicted in FIG. 1D; FIG. 6G The Cas12J polypeptide in Table 1 (or “Cas12J_3877103_16”) is also referred to herein as “CasΦ-4”;

[0125] (5) designated “Cas12J_10000002_47” or “Cas12J_1000002_112” and depicted in FIG. 1E; FIG. 6H The Cas12J polypeptide in Table 1 (or “Cas12J_1000002_112”) is also referred to herein as “CasΦ-5”;

[0126] (6) designated “Cas12J_10100763_4” and depicted in FIG. 1F; FIG. 6P The Cas12J polypeptide in Table 1 (or “Cas12J_10100763_4”) is also referred to herein as “CasΦ-6”;

[0127] (7) designated “Cas12J_1000007_143” or “Cas12J_1000001_267” and depicted in FIG. 1G; FIG. 6L The Cas12J polypeptide in Table 1 (or “Cas12J_1000001_267”) is also referred to herein as “CasΦ-7”;

[0128] (8) designated “Cas12J_10000286_53” and depicted in FIG. 1H (or “Cas12J_10000506_8” and depicted in FIG. 1I); FIG. 6O The Cas12J polypeptide in Table 1 (or “Cas12J_10000506_8”) is also referred to herein as “CasΦ-8”; FIG. 6M

[0129] ​(9) the Cas12J polypeptide designated "Cas12J_10001283_7" and depicted in FIG. 10J_10001283_7 FIG. 6E The Cas12J polypeptide designated "Cas12J_10001283_7" and depicted in FIG. 10J_10001283_7 is also referred to herein as "CasΦ-9";

[0130] (10) the Cas12J polypeptide designated "Cas12J_10037042_3" and depicted in FIG. 10J_10037042_3 FIG. 6A The Cas12J polypeptide designated "Cas12J_10037042_3" and depicted in FIG. 10J_10037042_3 is also referred to herein as "CasΦ-10".

[0131] In some cases, a Cas12J protein (of a composition and / or method of the application) comprises an amino acid sequence having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the Cas12J amino acid sequence designated "Cas12J_1947455" and depicted in FIG. 10J_1947455. For example, in some cases, a Cas12J protein comprises an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the Cas12J amino acid sequence designated "Cas12J_1947455" and depicted in FIG. 10J_1947455. In some cases, a Cas12J protein comprises an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the Cas12J amino acid sequence designated "Cas12J_1947455" and depicted in FIG. 10J_1947455. In some cases, a Cas12J protein comprises an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the Cas12J amino acid sequence designated "Cas12J_1947455" and depicted in FIG. 10J_1947455. In some cases, a Cas12J protein comprises an amino acid sequence having the Cas12J protein sequence designated "Cas12J_1947455" and depicted in FIG. 10J_1947455. In some cases, a Cas12J protein comprises an amino acid sequence having the Cas12J protein sequence designated "Cas12J_1947455" and depicted in FIG. 10J_1947455. FIG. 6A FIG. 6A FIG. 6A FIG. 6A FIG. 6A FIG. 6A ​​​​​The amino acid sequence of the Cas12J protein, except where the sequence includes amino acid substitutions (e.g., 1, 2, or 3 amino acid substitutions) that reduce the catalytic activity of the protein in its native form. In some cases, the Cas12J polypeptide has a length of 680 amino acids (aa) to 720 aa, such as 680 aa to 690 aa, 690 aa to 700 aa, 700 aa to 710 aa, or 710 aa to 720 aa. In some cases, the Cas12J polypeptide has a length of 707 amino acids. In some cases, the Cas12J polypeptide (e.g., containing amino acids described in...) is bound to... FIG. 9 The guide RNA of the Cas12J polypeptide having amino acid sequence identity of 20% or higher, 30% or higher, 40% or higher, 50% or higher, 60% or higher, 70% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% amino acid sequence identity includes the following nucleotide sequence: GTCTCGACTA ATCGAGCAAT CGTTTGAGAT CTCTCC (SEQ ID NO: 1) or its reverse complement. In some cases, the guide RNA comprises the nucleotide sequence (N)nGTCTCGACTAATCGAGCAA TCGTTTGAGA TCTCTCC (SEQ ID NO: 2) or its inverse complement, wherein N is any nucleotide and n is an integer from 15 to 30 (e.g., 15 to 20, 17 to 25, 17 to 22, 18 to 22, 18 to 20, 20 to 25, or 25 to 30). Cas12J_1947455 is specified (or...). FIG. 6A Cas12J_1947455_11) and depicted in FIG. 6B The Cas12J protein in this paper is also referred to as "ortholog #1" or "Cas12Φ-1".

[0132] In some cases, the Cas12J protein (of the compositions and / or methods of the present invention) comprises the protein described in FIG. 6B The document specifies that the Cas12J amino acid sequence of “Cas12J_071242” has 20% or higher sequence identity (e.g., 30% or higher, 40% or higher, 50% or higher, 60% or higher, 70% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity). For example, in some cases, the Cas12J protein includes amino acids that are described in [the document / reference needed]. FIG. 6BThe Cas12J amino acid sequence in the diagram has 50% or higher sequence identity (e.g., 60% or higher, 70% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity). In some cases, the Cas12J protein includes amino acids that are described in the diagram. FIG. 6B The Cas12J amino acid sequence in the literature has 80% or higher sequence identity (e.g., 85% or higher, 90% or higher, 95% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity). In some cases, the Cas12J protein includes amino acids that are similar to those described in the literature. FIG. 6B The Cas12J amino acid sequence has 90% or higher sequence identity (e.g., 95% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity). In some cases, the Cas12J protein includes amino acids depicted in... FIG. 6B The amino acid sequence of the Cas12J protein. In some cases, the Cas12J protein includes amino acids depicted in [the original text]. FIG. 6B The amino acid sequence of the Cas12J protein, except where the sequence includes amino acid substitutions (e.g., 1, 2, or 3 amino acid substitutions) that reduce the catalytic activity of the protein in its native form. In some cases, the Cas12J polypeptide has a length of 740 to 780 amino acids (aa), such as 740 to 750 aa, 750 to 760 aa, 760 to 770 aa, or 770 to 780 aa. In some cases, the Cas12J polypeptide has a length of 757 amino acids. In some cases, the Cas12J polypeptide is bound to (e.g., contained in the sequence described in...) FIG. 6BThe guide RNA of a Cas12J polypeptide having an amino acid sequence that is 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% amino acid sequence identity to the Cas12J amino acid sequence depicted in FIG. 1A includes the following nucleotide sequence: GTCGGAACGCTCAACGATTGCCCCTCACGAGGGGAC (SEQ ID NO: 3) or the reverse complement thereof. In some cases, the guide RNA comprises the nucleotide sequence (N)nGTCGGAACGCTCAACGATTGCCCCTCACGAGGGGAC (SEQ ID NO: 4) or the reverse complement thereof, wherein N is any nucleotide and n is an integer from 15 to 30 (e.g., 15 to 20, 17 to 25, 17 to 22, 18 to 22, 18 to 20, 20 to 25, or 25 to 30). The Cas12J protein depicted in FIG. 1A is designated Cas12J_2071242 and is also referred to herein as “ortholog #2” or “Cas12Φ-2.” FIG. 6C The Cas12J protein depicted in FIG. 1A is also referred to herein as “ortholog #2” or “Cas12Φ-2.”

[0133] In some cases, the Cas12J protein (of the present compositions and / or methods) comprises an amino acid sequence that is 20% or more (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) sequence identity to the Cas12J amino acid sequence depicted in FIG. 1A and designated “Cas12J_1973640.” FIG. 6C In some cases, the Cas12J protein (of the present compositions and / or methods) comprises an amino acid sequence that is 20% or more (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) sequence identity to the Cas12J amino acid sequence depicted in FIG. 1A and designated “Cas12J_1973640.” FIG. 6C In some cases, the Cas12J protein (of the present compositions and / or methods) comprises an amino acid sequence that is 20% or more (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) sequence identity to the Cas12J amino acid sequence depicted in FIG. 1A and designated “Cas12J_1973640.” FIG. 6C In some cases, the Cas12J protein (of the present compositions and / or methods) comprises an amino acid sequence that is 20% or more (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) sequence identity to the Cas12J amino acid sequence depicted in FIG. 1A and designated “Cas12J_1973640.” FIG. 6CThe Cas12J amino acid sequence has 90% or higher sequence identity (e.g., 95% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity). In some cases, the Cas12J protein includes amino acids depicted in... FIG. 6C The amino acid sequence of the Cas12J protein. In some cases, the Cas12J protein includes amino acids depicted in [the original text]. FIG. 6D The amino acid sequence of the Cas12J protein, except where the sequence includes amino acid substitutions (e.g., 1, 2, or 3 amino acid substitutions) that reduce the catalytic activity of the protein in its native form. In some cases, the Cas12J polypeptide has a length of 740 to 780 amino acids (aa), such as 740 to 750, 750 to 760, 760 to 770, or 770 to 780 aa. In some cases, the Cas12J polypeptide has a length of 765 amino acids.

[0134] In some cases, the Cas12J protein (of the compositions and / or methods of the present invention) comprises the protein described in FIG. 6D The document specifies that the Cas12J amino acid sequence of “Cas12J_3339380” has 20% or higher sequence identity (e.g., 30% or higher, 40% or higher, 50% or higher, 60% or higher, 70% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity). For example, in some cases, the Cas12J protein includes amino acids that are described in [the document / reference]. FIG. 6D The Cas12J amino acid sequence in the diagram has 50% or higher sequence identity (e.g., 60% or higher, 70% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity). In some cases, the Cas12J protein includes amino acids that are described in the diagram. FIG. 6D The Cas12J amino acid sequence in the literature has 80% or higher sequence identity (e.g., 85% or higher, 90% or higher, 95% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity). In some cases, the Cas12J protein includes amino acids that are similar to those described in the literature. FIG. 6D The Cas12J amino acid sequence has 90% or higher sequence identity (e.g., 95% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity). In some cases, the Cas12J protein includes amino acids depicted in... FIG. 6DThe amino acid sequence of the Cas12J protein. In some cases, the Cas12J protein includes amino acids depicted in [the original text]. FIG. 6D The amino acid sequence of the Cas12J protein, except where the sequence includes amino acid substitutions (e.g., 1, 2, or 3 amino acid substitutions) that reduce the catalytic activity of the protein in its native form. In some cases, the Cas12J polypeptide has a length of 740 to 780 amino acids (aa), such as 740 to 750 aa, 750 to 760 aa, 760 to 770 aa, or 770 to 780 aa. In some cases, the Cas12J polypeptide has a length of 766 amino acids. In some cases, the Cas12J polypeptide (e.g., containing amino acids described in...) is bound to... FIG. 6D The guide RNA of the Cas12J polypeptide having 20% ​​or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% amino acid sequence identity includes the following nucleotide sequence: GTCCCAGCGT ACTGGGCAAT CAATAGTCGT TTTGGT (SEQ ID NO: 5) or its reverse complement. In some cases, the guide RNA comprises the nucleotide sequence (N)nGTCCCAGCGTACTGGGCAA TCAATAGTCG TTTTGGT (SEQ ID NO: 6) or its reverse complement, wherein N is any nucleotide and n is an integer from 15 to 30 (e.g., 15 to 20, 17 to 25, 17 to 22, 18 to 22, 18 to 20, 20 to 25, or 25 to 30). Cas12J_3339380 is specified and depicted in... FIG. 6E The Cas12J protein in this paper is also referred to as "ortholog #3" or "Cas12Φ-3".

[0135] In some cases, the Cas12J protein (of the compositions and / or methods of the present invention) comprises the protein described in FIG. 6E The document specifies that the Cas12J amino acid sequence of “Cas12J_10037042_3” has 20% or higher sequence identity (e.g., 30% or higher, 40% or higher, 50% or higher, 60% or higher, 70% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity). For example, in some cases, the Cas12J protein includes amino acids that are described in [the document / reference needed]. FIG. 6Ethe Cas12J amino acid sequence depicted in SEQ ID NO: 10020921_9 has 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the amino acid sequence of a Cas12J amino acid sequence in Table 2. In some cases, a Cas12J protein comprises an amino acid sequence having 20% or more sequence identity to the Cas12J amino acid sequence depicted in SEQ ID NO: 10020921_9, except that the sequence includes one or more amino acid substitutions that reduce the catalytic activity of the protein. In some cases, a Cas12J polypeptide has a length of 780 amino acids (aa) to 820 aa, e.g., 780 aa to 790 aa, 790 aa to 800 aa, 800 aa to 810 aa, or 810 aa to 820 aa. In some cases, a Cas12J polypeptide has a length of 812 amino acids. FIG. 6E the Cas12J amino acid sequence depicted in SEQ ID NO: 10020921_9 has 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the amino acid sequence of a Cas12J amino acid sequence in Table 2. In some cases, a Cas12J protein comprises an amino acid sequence having 80% or more sequence identity to the Cas12J amino acid sequence depicted in SEQ ID NO: 10020921_9, except that the sequence includes one or more amino acid substitutions that reduce the catalytic activity of the protein. In some cases, a Cas12J polypeptide has a length of 780 amino acids (aa) to 820 aa, e.g., 780 aa to 790 aa, 790 aa to 800 aa, 800 aa to 810 aa, or 810 aa to 820 aa. In some cases, a Cas12J polypeptide has a length of 812 amino acids. FIG. 6E the Cas12J amino acid sequence depicted in SEQ ID NO: 10020921_9 has 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the amino acid sequence of a Cas12J amino acid sequence in Table 2. In some cases, a Cas12J protein comprises an amino acid sequence having 90% or more sequence identity to the Cas12J amino acid sequence depicted in SEQ ID NO: 10020921_9, except that the sequence includes one or more amino acid substitutions that reduce the catalytic activity of the protein. In some cases, a Cas12J polypeptide has a length of 780 amino acids (aa) to 820 aa, e.g., 780 aa to 790 aa, 790 aa to 800 aa, 800 aa to 810 aa, or 810 aa to 820 aa. In some cases, a Cas12J polypeptide has a length of 812 amino acids. FIG. 6E the Cas12J amino acid sequence depicted in SEQ ID NO: 10020921_9 has 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the amino acid sequence of a Cas12J amino acid sequence in Table 2. In some cases, a Cas12J protein comprises an amino acid sequence having 90% or more sequence identity to the Cas12J amino acid sequence depicted in SEQ ID NO: 10020921_9, except that the sequence includes one or more amino acid substitutions that reduce the catalytic activity of the protein. In some cases, a Cas12J polypeptide has a length of 780 amino acids (aa) to 820 aa, e.g., 780 aa to 790 aa, 790 aa to 800 aa, 800 aa to 810 aa, or 810 aa to 820 aa. In some cases, a Cas12J polypeptide has a length of 812 amino acids. FIG. 6F the Cas12J amino acid sequence depicted in SEQ ID NO: 10020921_9 has 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the amino acid sequence of a Cas12J amino acid sequence in Table 2. In some cases, a Cas12J protein comprises an amino acid sequence having 90% or more sequence identity to the Cas12J amino acid sequence depicted in SEQ ID NO: 10020921_9, except that the sequence includes one or more amino acid substitutions that reduce the catalytic activity of the protein. In some cases, a Cas12J polypeptide has a length of 780 amino acids (aa) to 820 aa, e.g., 780 aa to 790 aa, 790 aa to 800 aa, 800 aa to 810 aa, or 810 aa to 820 aa. In some cases, a Cas12J polypeptide has a length of 812 amino acids.

[0136] In some cases, a Cas12J protein (of a composition and / or method of the application) comprises an amino acid sequence having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the Cas12J amino acid sequence depicted in SEQ ID NO: 10020921_9. For example, in some cases, a Cas12J protein comprises an amino acid sequence having 20% or more sequence identity to the Cas12J amino acid sequence depicted in SEQ ID NO: 10020921_9, except that the sequence includes one or more amino acid substitutions that reduce the catalytic activity of the protein. FIG. 6F the Cas12J amino acid sequence depicted in SEQ ID NO: 10020921_9 has 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the amino acid sequence of a Cas12J amino acid sequence in Table 2. In some cases, a Cas12J protein comprises an amino acid sequence having 20% or more sequence identity to the Cas12J amino acid sequence depicted in SEQ ID NO: 10020921_9, except that the sequence includes one or more amino acid substitutions that reduce the catalytic activity of the protein. In some cases, a Cas12J polypeptide has a length of 780 amino acids (aa) to 820 aa, e.g., 780 aa to 790 aa, 790 aa to 800 aa, 800 aa to 810 aa, or 810 aa to 820 aa. In some cases, a Cas12J polypeptide has a length of 812 amino acids. FIG. 6Fthe Cas12J amino acid sequence depicted in SEQ ID NO: 10000002_47 has 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the amino acid sequence of a Cas12J amino acid sequence in Table 1. In some cases, a Cas12J protein includes an amino acid sequence having 20% or more sequence identity to the Cas12J amino acid sequence depicted in SEQ ID NO: 10000002_47, except that the sequence includes one or more amino acid substitutions that reduce the naturally occurring catalytic activity of the protein. In some cases, a Cas12J polypeptide has a length of 812 amino acids. FIG. 6F the Cas12J amino acid sequence depicted in SEQ ID NO: 10000002_47 has 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the amino acid sequence of a Cas12J amino acid sequence in Table 1. In some cases, a Cas12J protein includes an amino acid sequence having 20% or more sequence identity to the Cas12J amino acid sequence depicted in SEQ ID NO: 10000002_47, except that the sequence includes one or more amino acid substitutions that reduce the naturally occurring catalytic activity of the protein. In some cases, a Cas12J polypeptide has a length of 812 amino acids. FIG. 6F the Cas12J amino acid sequence depicted in SEQ ID NO: 10000002_47 has 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the amino acid sequence of a Cas12J amino acid sequence in Table 1. In some cases, a Cas12J protein includes an amino acid sequence having 20% or more sequence identity to the Cas12J amino acid sequence depicted in SEQ ID NO: 10000002_47, except that the sequence includes one or more amino acid substitutions that reduce the naturally occurring catalytic activity of the protein. In some cases, a Cas12J polypeptide has a length of 812 amino acids. FIG. 6F the Cas12J amino acid sequence depicted in SEQ ID NO: 10000002_47 has 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the amino acid sequence of a Cas12J amino acid sequence in Table 1. In some cases, a Cas12J protein includes an amino acid sequence having 20% or more sequence identity to the Cas12J amino acid sequence depicted in SEQ ID NO: 10000002_47, except that the sequence includes one or more amino acid substitutions that reduce the naturally occurring catalytic activity of the protein. In some cases, a Cas12J polypeptide has a length of 812 amino acids. FIG. 6G the Cas12J amino acid sequence depicted in SEQ ID NO: 10000002_47 has 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the amino acid sequence of a Cas12J amino acid sequence in Table 1. In some cases, a Cas12J protein includes an amino acid sequence having 20% or more sequence identity to the Cas12J amino acid sequence depicted in SEQ ID NO: 10000002_47, except that the sequence includes one or more amino acid substitutions that reduce the naturally occurring catalytic activity of the protein. In some cases, a Cas12J polypeptide has a length of 812 amino acids.

[0137] the Cas12J amino acid sequence depicted in SEQ ID NO: 10000002_47 has 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the amino acid sequence of a Cas12J amino acid sequence in Table 1. In some cases, a Cas12J protein includes an amino acid sequence having 20% or more sequence identity to the Cas12J amino acid sequence depicted in SEQ ID NO: 10000002_47, except that the sequence includes one or more amino acid substitutions that reduce the naturally occurring catalytic activity of the protein. In some cases, a Cas12J polypeptide has a length of 812 amino acids. FIG. 6G the Cas12J amino acid sequence depicted in SEQ ID NO: 10000002_47 has 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the amino acid sequence of a Cas12J amino acid sequence in Table 1. In some cases, a Cas12J protein includes an amino acid sequence having 20% or more sequence identity to the Cas12J amino acid sequence depicted in SEQ ID NO: 10000002_47, except that the sequence includes one or more amino acid substitutions that reduce the naturally occurring catalytic activity of the protein. In some cases, a Cas12J polypeptide has a length of 812 amino acids. FIG. 6GThe Cas12J amino acid sequence in the diagram has 50% or higher sequence identity (e.g., 60% or higher, 70% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity). In some cases, the Cas12J protein includes amino acids that are described in the diagram. FIG. 6G The Cas12J amino acid sequence in the literature has 80% or higher sequence identity (e.g., 85% or higher, 90% or higher, 95% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity). In some cases, the Cas12J protein includes amino acids that are similar to those described in the literature. FIG. 6G The Cas12J amino acid sequence has 90% or higher sequence identity (e.g., 95% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity). In some cases, the Cas12J protein includes amino acids depicted in... FIG. 6G The amino acid sequence of the Cas12J protein. In some cases, the Cas12J protein includes amino acids depicted in [the original text]. FIG. 6G The amino acid sequence of the Cas12J protein, except where the sequence includes amino acid substitutions (e.g., 1, 2, or 3 amino acid substitutions) that reduce the catalytic activity of the protein in its native form. In some cases, the Cas12J polypeptide has a length of 770 amino acids (aa) to 810 aa, such as 770 aa to 780 aa, 780 aa to 790 aa, 790 aa to 800 aa, or 800 aa to 810 aa. In some cases, the Cas12J polypeptide has a length of 793 amino acids. In some cases, the Cas12J polypeptide (e.g., containing amino acids described in...) is bound to... FIG. 6HThe guide RNA of the Cas12J polypeptide having amino acid sequence identity of 20% or higher, 30% or higher, 40% or higher, 50% or higher, 60% or higher, 70% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% amino acid sequence identity includes the following nucleotide sequence: GGATCCAATC CTTTTTGATT GCCCAATTCGTTGGGAC (SEQ ID NO: 7) or its reverse complement. In some cases, the guide RNA comprises the nucleotide sequence (N)nGGATCCAAT CCTTTTTGAT TGCCCAATTC GTTGGGAC (SEQ ID NO: 8) or its reverse complement, wherein N is any nucleotide and n is an integer from 15 to 30 (e.g., 15 to 20, 17 to 25, 17 to 22, 18 to 22, 18 to 20, 20 to 25, or 25 to 30).

[0138] In some cases, the Cas12J protein (of the compositions and / or methods of the present invention) comprises the protein described in FIG. 6H The document specifies that the Cas12J amino acid sequence of “Cas12J_10100763_4” has 20% or higher sequence identity (e.g., 30% or higher, 40% or higher, 50% or higher, 60% or higher, 70% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity). For example, in some cases, the Cas12J protein includes amino acids that are described in [the document / reference]. FIG. 6H The Cas12J amino acid sequence in the diagram has 50% or higher sequence identity (e.g., 60% or higher, 70% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity). In some cases, the Cas12J protein includes amino acids that are described in the diagram. FIG. 6H The Cas12J amino acid sequence in the literature has 80% or higher sequence identity (e.g., 85% or higher, 90% or higher, 95% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity). In some cases, the Cas12J protein includes amino acids that are similar to those described in the literature. FIG. 6H The Cas12J amino acid sequence has 90% or higher sequence identity (e.g., 95% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity). In some cases, the Cas12J protein includes amino acids depicted in... FIG. 6Hthe amino acid sequence of the Cas12J protein sequence depicted in FIG. 6I the amino acid sequence of the Cas12J protein sequence depicted in, except that the sequence includes an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein. In some cases, a Cas12J polypeptide has a length of 420 amino acids (aa) to 460 aa, e.g., 420 aa to 430 aa, 430 aa to 440 aa, 440 aa to 450 aa, or 450 aa to 460 aa. In some cases, a Cas12J polypeptide has a length of 441 amino acids.

[0139] In some cases, a Cas12J protein (of a composition and / or method of the application) includes an amino acid sequence having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the Cas12J amino acid sequence depicted in FIG. 6I In some cases, a Cas12J protein (of a composition and / or method of the application) includes an amino acid sequence having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the Cas12J amino acid sequence depicted in FIG. 6I In some cases, a Cas12J protein (of a composition and / or method of the application) includes an amino acid sequence having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the Cas12J amino acid sequence depicted in FIG. 6I In some cases, a Cas12J protein (of a composition and / or method of the application) includes an amino acid sequence having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the Cas12J amino acid sequence depicted in FIG. 6I In some cases, a Cas12J protein (of a composition and / or method of the application) includes an amino acid sequence having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the Cas12J amino acid sequence depicted in FIG. 6I In some cases, a Cas12J protein (of a composition and / or method of the application) includes an amino acid sequence having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the Cas12J amino acid sequence depicted in FIG. 6JThe amino acid sequence of the Cas12J protein, with the exception that the sequence includes amino acid substitutions (e.g., 1, 2, or 3 amino acid substitutions) that reduce the catalytic activity of the protein in its native form. In some cases, the Cas12J polypeptide has a length of 790 to 830 amino acids (aa), for example, 790 to 800 aa, 800 to 810 aa, 810 to 820 aa, or 820 to 830 aa. In some cases, the Cas12J polypeptide has a length of 812 amino acids.

[0140] In some cases, the Cas12J protein (of the compositions and / or methods of the present invention) comprises the protein described in FIG. 6J The document specifies that the Cas12J amino acid sequence of “Cas12J_10000724_71” has 20% or higher sequence identity (e.g., 30% or higher, 40% or higher, 50% or higher, 60% or higher, 70% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity). For example, in some cases, the Cas12J protein includes amino acids that are described in [the document / reference needed]. FIG. 6J The Cas12J amino acid sequence in the diagram has 50% or higher sequence identity (e.g., 60% or higher, 70% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity). In some cases, the Cas12J protein includes amino acids that are described in the diagram. FIG. 6J The Cas12J amino acid sequence in the literature has 80% or higher sequence identity (e.g., 85% or higher, 90% or higher, 95% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity). In some cases, the Cas12J protein includes amino acids that are similar to those described in the literature. FIG. 6J The Cas12J amino acid sequence has 90% or higher sequence identity (e.g., 95% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity). In some cases, the Cas12J protein includes amino acids depicted in... FIG. 6J The amino acid sequence of the Cas12J protein. In some cases, the Cas12J protein includes amino acids depicted in [the original text]. FIG. 6Jthe Cas12J amino acid sequence depicted in FIG. 1A, except that the sequence includes an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein. In some cases, a Cas12J polypeptide has a length of 790 amino acids (aa) to 830 aa, e.g., 790 aa to 800 aa, 800 aa to 810 aa, 810 aa to 820 aa, or 820 aa to 830 aa. In some cases, a Cas12J polypeptide has a length of 812 amino acids. In some cases, a guide RNA for a Cas12J polypeptide (e.g., comprising an amino acid sequence having 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% amino acid sequence identity to the Cas12J amino acid sequence depicted in FIG. 1A) comprises the following nucleotide sequence: GGATCTGAGG ATCATTATTG CTCGTTACGACGAGAC (SEQ ID NO: 9) or the reverse complement thereof. In some cases, the guide RNA comprises the nucleotide sequence (N)nGGATCTGAG GATCATTATT GCTCGTTACG ACGAGAC (SEQ ID NO: 10) or the reverse complement thereof, wherein N is any nucleotide and n is an integer from 15 to 30 (e.g., 15 to 20, 17 to 25, 17 to 22, 18 to 22, 18 to 20, 20 to 25, or 25 to 30). FIG. 6J the Cas12J amino acid sequence depicted in FIG. 1A, except that the sequence includes an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein. In some cases, a Cas12J polypeptide has a length of 790 amino acids (aa) to 830 aa, e.g., 790 aa to 800 aa, 800 aa to 810 aa, 810 aa to 820 aa, or 820 aa to 830 aa. In some cases, a Cas12J polypeptide has a length of 812 amino acids. In some cases, a guide RNA for a Cas12J polypeptide (e.g., comprising an amino acid sequence having 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% amino acid sequence identity to the Cas12J amino acid sequence depicted in FIG. 1A) comprises the following nucleotide sequence: GGATCTGAGG ATCATTATTG CTCGTTACGACGAGAC (SEQ ID NO: 9) or the reverse complement thereof. In some cases, the guide RNA comprises the nucleotide sequence (N)nGGATCTGAG GATCATTATT GCTCGTTACG ACGAGAC (SEQ ID NO: 10) or the reverse complement thereof, wherein N is any nucleotide and n is an integer from 15 to 30 (e.g., 15 to 20, 17 to 25, 17 to 22, 18 to 22, 18 to 20, 20 to 25, or 25 to 30). FIG. 6K the Cas12J amino acid sequence depicted in FIG. 1A, except that the sequence includes an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein. In some cases, a Cas12J polypeptide has a length of 790 amino acids (aa) to 830 aa, e.g., 790 aa to 800 aa, 800 aa to 810 aa, 810 aa to 820 aa, or 820 aa to 830 aa. In some cases, a Cas12J polypeptide has a length of 812 amino acids. In some cases, a guide RNA for a Cas12J polypeptide (e.g., comprising an amino acid sequence having 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% amino acid sequence identity to the Cas12J amino acid sequence depicted in FIG. 1A) comprises the following nucleotide sequence: GGATCTGAGG ATCATTATTG CTCGTTACGACGAGAC (SEQ ID NO: 9) or the reverse complement thereof. In some cases, the guide RNA comprises the nucleotide sequence (N)nGGATCTGAG GATCATTATT GCTCGTTACG ACGAGAC (SEQ ID NO: 10) or the reverse complement thereof, wherein N is any nucleotide and n is an integer from 15 to 30 (e.g., 15 to 20, 17 to 25, 17 to 22, 18 to 22, 18 to 20, 20 to 25, or 25 to 30).

[0141] In some cases, the Cas12J protein (of the compositions and / or methods of the present invention) comprises the protein described in FIG. 6K The document specifies that the Cas12J amino acid sequence of “Cas12J_1000001_267” has 20% or higher sequence identity (e.g., 30% or higher, 40% or higher, 50% or higher, 60% or higher, 70% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity). For example, in some cases, the Cas12J protein includes amino acids that are described in [the document / concept]. FIG. 6K The Cas12J amino acid sequence in the diagram has 50% or higher sequence identity (e.g., 60% or higher, 70% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity). In some cases, the Cas12J protein includes amino acids that are described in the diagram. FIG. 6K The Cas12J amino acid sequence in the literature has 80% or higher sequence identity (e.g., 85% or higher, 90% or higher, 95% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity). In some cases, the Cas12J protein includes amino acids that are similar to those described in the literature. FIG. 6K The Cas12J amino acid sequence has 90% or higher sequence identity (e.g., 95% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity). In some cases, the Cas12J protein includes amino acids depicted in... FIG. 6K The amino acid sequence of the Cas12J protein. In some cases, the Cas12J protein includes amino acids depicted in [the original text]. FIG. 6K The amino acid sequence of the Cas12J protein, except where the sequence includes amino acid substitutions (e.g., 1, 2, or 3 amino acid substitutions) that reduce the catalytic activity of the protein in its native form. In some cases, the Cas12J polypeptide has a length of 750 to 790 amino acids (aa), such as 750 to 760 aa, 760 to 770 aa, 770 to 780 aa, or 780 to 790 aa. In some cases, the Cas12J polypeptide has a length of 772 amino acids. In some cases, the Cas12J polypeptide (e.g., containing amino acids described in...) is bound to... FIG. 6LThe guide RNA of the Cas12J polypeptide having 20% ​​or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% amino acid sequence identity includes the following nucleotide sequence: GTCTCAGCGT ACTGAGCAAT CAAAAGGTTTCGCAGG (SEQ ID NO: 13) or its reverse complement. In some cases, the guide RNA comprises the nucleotide sequence (N)nGTCTCAGCG TACTGAGCAA TCAAAAGGTT TCGCAGG (SEQ ID NO: 14) or its reverse complement, wherein N is any nucleotide and n is an integer from 15 to 30 (e.g., 15 to 20, 17 to 25, 17 to 22, 18 to 22, 18 to 20, 20 to 25, or 25 to 30).

[0142] In some cases, the Cas12J protein (of the compositions and / or methods of the present invention) comprises the protein described in FIG. 6L The document specifies that the Cas12J amino acid sequence of “Cas12J_10000286_53” has 20% or higher sequence identity (e.g., 30% or higher, 40% or higher, 50% or higher, 60% or higher, 70% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity). For example, in some cases, the Cas12J protein includes amino acids that are described in [the document / reference]. FIG. 6L The Cas12J amino acid sequence in the diagram has 50% or higher sequence identity (e.g., 60% or higher, 70% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity). In some cases, the Cas12J protein includes amino acids that are described in the diagram. FIG. 6L The Cas12J amino acid sequence in the literature has 80% or higher sequence identity (e.g., 85% or higher, 90% or higher, 95% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity). In some cases, the Cas12J protein includes amino acids that are similar to those described in the literature. FIG. 6L The Cas12J amino acid sequence has 90% or higher sequence identity (e.g., 95% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity). In some cases, the Cas12J protein includes amino acids depicted in... FIG. 6LThe amino acid sequence of the Cas12J protein. In some cases, the Cas12J protein includes amino acids depicted in [the original text]. FIG. 6L The amino acid sequence of the Cas12J protein, except where the sequence includes amino acid substitutions (e.g., 1, 2, or 3 amino acid substitutions) that reduce the catalytic activity of the protein in its native form. In some cases, the Cas12J polypeptide has a length of 700 amino acids (aa) to 740 aa, such as 700 aa to 710 aa, 710 aa to 720 aa, 720 aa to 730 aa, or 730 aa to 740 aa. In some cases, the Cas12J polypeptide has a length of 717 amino acids. In some cases, the Cas12J polypeptide is bound to (e.g., contained in the sequence described in...) FIG. 6M The guide RNA of the Cas12J polypeptide having 20% ​​or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% amino acid sequence identity includes the following nucleotide sequence: GTCTCCTCGT AAGGAGCAAT CTATTAGTCTTGAAAG (SEQ ID NO: 15) or its reverse complement. In some cases, the guide RNA comprises the nucleotide sequence (N)nGTCTCCTCG TAAGGAGCAA TCTATTAGTC TTGAAAG (SEQ ID NO: 16) or its reverse complement, wherein N is any nucleotide and n is an integer from 15 to 30 (e.g., 15 to 20, 17 to 25, 17 to 22, 18 to 22, 18 to 20, 20 to 25, or 25 to 30).

[0143] In some cases, the Cas12J protein (of the compositions and / or methods of the present invention) comprises the protein described in FIG. 6M The document specifies that the Cas12J amino acid sequence of “Cas12J_10001283_7” has 20% or higher sequence identity (e.g., 30% or higher, 40% or higher, 50% or higher, 60% or higher, 70% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity). For example, in some cases, the Cas12J protein includes amino acids that are described in [the document / concept]. FIG. 6MThe Cas12J amino acid sequence in the diagram has 50% or higher sequence identity (e.g., 60% or higher, 70% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity). In some cases, the Cas12J protein includes amino acids that are described in the diagram. FIG. 6M The Cas12J amino acid sequence in the literature has 80% or higher sequence identity (e.g., 85% or higher, 90% or higher, 95% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity). In some cases, the Cas12J protein includes amino acids that are similar to those described in the literature. FIG. 6M The Cas12J amino acid sequence has 90% or higher sequence identity (e.g., 95% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity). In some cases, the Cas12J protein includes amino acids depicted in... FIG. 6M The amino acid sequence of the Cas12J protein. In some cases, the Cas12J protein includes amino acids depicted in [the original text]. FIG. 6M The amino acid sequence of the Cas12J protein, except where the sequence includes amino acid substitutions (e.g., 1, 2, or 3 amino acid substitutions) that reduce the catalytic activity of the protein in its native form. In some cases, the Cas12J polypeptide has a length of 770 amino acids (aa) to 810 aa, such as 770 aa to 780 aa, 780 aa to 790 aa, 790 aa to 800 aa, or 800 aa to 810 aa. In some cases, the Cas12J polypeptide has a length of 793 amino acids. In some cases, the Cas12J polypeptide (e.g., containing amino acids described in...) is bound to... FIG. 6NThe guide RNA of a Cas12J polypeptide having an amino acid sequence that is 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% amino acid sequence identity to the Cas12J amino acid sequence depicted in FIG. 1A includes the following nucleotide sequence: GTCTCGGCGC ACCGAGCAAT CAGCGAGGTCTTCTAC (SEQ ID NO: 17) or the reverse complement thereof. In some cases, the guide RNA comprises the nucleotide sequence (N)nGTCTCGGCGC ACCGAGCAAT CAGCGAGGTCTTCTAC (SEQ ID NO: 18) or the reverse complement thereof, wherein N is any nucleotide and n is an integer from 15 to 30 (e.g., 15 to 20, 17 to 25, 17 to 22, 18 to 22, 18 to 20, 20 to 25, or 25 to 30).

[0144] In some cases, the Cas12J protein (of the present compositions and / or methods) comprises an amino acid sequence that is 20% or more (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) sequence identity to the Cas12J amino acid sequence depicted in FIG. 1A and designated “Cas12J_1000002_112.” For example, in some cases, the Cas12J protein comprises an amino acid sequence that is 50% or more (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) sequence identity to the Cas12J amino acid sequence depicted in FIG. 1A and designated “Cas12J_1000002_112.” FIG. 6N In some cases, the Cas12J protein (of the present compositions and / or methods) comprises an amino acid sequence that is 20% or more (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) sequence identity to the Cas12J amino acid sequence depicted in FIG. 1A and designated “Cas12J_1000002_112.” For example, in some cases, the Cas12J protein comprises an amino acid sequence that is 50% or more (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) sequence identity to the Cas12J amino acid sequence depicted in FIG. 1A and designated “Cas12J_1000002_112.” FIG. 6N In some cases, the Cas12J protein (of the present compositions and / or methods) comprises an amino acid sequence that is 20% or more (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) sequence identity to the Cas12J amino acid sequence depicted in FIG. 1A and designated “Cas12J_1000002_112.” For example, in some cases, the Cas12J protein comprises an amino acid sequence that is 50% or more (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) sequence identity to the Cas12J amino acid sequence depicted in FIG. 1A and designated “Cas12J_1000002_112.” FIG. 6N In some cases, the Cas12J protein (of the present compositions and / or methods) comprises an amino acid sequence that is 20% or more (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) sequence identity to the Cas12J amino acid sequence depicted in FIG. 1A and designated “Cas12J_1000002_112.” For example, in some cases, the Cas12J protein comprises an amino acid sequence that is 50% or more (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) sequence identity to the Cas12J amino acid sequence depicted in FIG. 1A and designated “Cas12J_1000002_112.” FIG. 6N In some cases, the Cas12J protein (of the present compositions and / or methods) comprises an amino acid sequence that is 20% or more (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) sequence identity to the Cas12J amino acid sequence depicted in FIG. 1A and designated “Cas12J_1000002_112.” For example, in some cases, the Cas12J protein comprises an amino acid sequence that is 50% or more (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) sequence identity to the Cas12J amino acid sequence depicted in FIG. 1A and designated “Cas12J_1000002_112.” FIG. 6NThe amino acid sequence of the Cas12J protein. In some cases, the Cas12J protein includes amino acids depicted in [the original text]. FIG. 6N The amino acid sequence of the Cas12J protein, except where the sequence includes amino acid substitutions (e.g., 1, 2, or 3 amino acid substitutions) that reduce the catalytic activity of the protein in its native form. In some cases, the Cas12J polypeptide has a length of 770 amino acids (aa) to 810 aa, such as 770 aa to 780 aa, 780 aa to 790 aa, 790 aa to 800 aa, or 800 aa to 810 aa. In some cases, the Cas12J polypeptide has a length of 793 amino acids. In some cases, the Cas12J polypeptide (e.g., containing amino acids described in...) is bound to... FIG. 6O The guide RNA of the Cas12J polypeptide having 20% ​​or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% amino acid sequence identity includes the following nucleotide sequence: GTCCCAACGA ATTGGGCAAT CAAAAAGGATTGGATCC (SEQ ID NO: 19) or its reverse complement. In some cases, the guide RNA comprises the nucleotide sequence (N)nGTCCCAACG AATTGGGCAA TCAAAAAGGA TTGGATCC (SEQ ID NO: 20) or its reverse complement, wherein N is any nucleotide and n is an integer from 15 to 30 (e.g., 15 to 20, 17 to 25, 17 to 22, 18 to 22, 18 to 20, 20 to 25, or 25 to 30).

[0145] In some cases, the Cas12J protein (of the compositions and / or methods of the present invention) comprises the protein described in FIG. 6O The document specifies that the Cas12J amino acid sequence of “Cas12J_10000506_8” has 20% or higher sequence identity (e.g., 30% or higher, 40% or higher, 50% or higher, 60% or higher, 70% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity). For example, in some cases, the Cas12J protein includes amino acids that are described in [the document / reference]. FIG. 6OThe Cas12J amino acid sequence in the diagram has 50% or higher sequence identity (e.g., 60% or higher, 70% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity). In some cases, the Cas12J protein includes amino acids that are described in the diagram. FIG. 6O The Cas12J amino acid sequence in the literature has 80% or higher sequence identity (e.g., 85% or higher, 90% or higher, 95% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity). In some cases, the Cas12J protein includes amino acids that are similar to those described in the literature. FIG. 6O The Cas12J amino acid sequence has 90% or higher sequence identity (e.g., 95% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity). In some cases, the Cas12J protein includes amino acids depicted in... FIG. 6O The amino acid sequence of the Cas12J protein. In some cases, the Cas12J protein includes amino acids depicted in [the original text]. FIG. 6O The amino acid sequence of the Cas12J protein, except where the sequence includes amino acid substitutions (e.g., 1, 2, or 3 amino acid substitutions) that reduce the catalytic activity of the protein in its native form. In some cases, the Cas12J polypeptide has a length of 700 amino acids (aa) to 740 aa, such as 700 aa to 710 aa, 710 aa to 720 aa, 720 aa to 730 aa, or 730 aa to 740 aa. In some cases, the Cas12J polypeptide has a length of 717 amino acids. In some cases, the Cas12J polypeptide is bound to (e.g., contained in the sequence described in...) FIG. 6PThe guide RNA of a Cas12J polypeptide having an amino acid sequence that is 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% amino acid sequence identity to the Cas12J amino acid sequence depicted in FIG. 1A includes the following nucleotide sequence: GTCTCCTCGTAAGGAGCAATCTATTAGTCTTGAAAG (SEQ ID NO: 15) or the reverse complement thereof. In some cases, the guide RNA comprises the nucleotide sequence (N)nGTCTCCTCGTAAGGAGCAATCTATTAGTCTTGAAAG (SEQ ID NO: 16) or the reverse complement thereof, wherein N is any nucleotide and n is an integer from 15 to 30 (e.g., 15 to 20, 17 to 25, 17 to 22, 18 to 22, 18 to 20, 20 to 25, or 25 to 30).

[0146] In some cases, the Cas12J protein (of the present compositions and / or methods) comprises an amino acid sequence that is 20% or more (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) sequence identity to the Cas12J amino acid sequence depicted in FIG. 1A and designated “Cas12J_1000007_143.” For example, in some cases, the Cas12J protein comprises an amino acid sequence that is 50% or more (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) sequence identity to the Cas12J amino acid sequence depicted in FIG. 1A and designated “Cas12J_1000007_143.” FIG. 6P In some cases, the Cas12J protein (of the present compositions and / or methods) comprises an amino acid sequence that is 20% or more (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) sequence identity to the Cas12J amino acid sequence depicted in FIG. 1A and designated “Cas12J_1000007_143.” For example, in some cases, the Cas12J protein comprises an amino acid sequence that is 50% or more (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) sequence identity to the Cas12J amino acid sequence depicted in FIG. 1A and designated “Cas12J_1000007_143.” FIG. 6P In some cases, the Cas12J protein (of the present compositions and / or methods) comprises an amino acid sequence that is 20% or more (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) sequence identity to the Cas12J amino acid sequence depicted in FIG. 1A and designated “Cas12J_1000007_143.” For example, in some cases, the Cas12J protein comprises an amino acid sequence that is 50% or more (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) sequence identity to the Cas12J amino acid sequence depicted in FIG. 1A and designated “Cas12J_1000007_143.” FIG. 6P In some cases, the Cas12J protein (of the present compositions and / or methods) comprises an amino acid sequence that is 20% or more (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) sequence identity to the Cas12J amino acid sequence depicted in FIG. 1A and designated “Cas12J_1000007_143.” For example, in some cases, the Cas12J protein comprises an amino acid sequence that is 50% or more (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) sequence identity to the Cas12J amino acid sequence depicted in FIG. 1A and designated “Cas12J_1000007_143.” FIG. 6P In some cases, the Cas12J protein (of the present compositions and / or methods) comprises an amino acid sequence that is 20% or more (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) sequence identity to the Cas12J amino acid sequence depicted in FIG. 1A and designated “Cas12J_1000007_143.” For example, in some cases, the Cas12J protein comprises an amino acid sequence that is 50% or more (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) sequence identity to the Cas12J amino acid sequence depicted in FIG. 1A and designated “Cas12J_1000007_143.” FIG. 6PThe amino acid sequence of the Cas12J protein. In some cases, the Cas12J protein includes amino acids depicted in [the original text]. FIG. 6P The amino acid sequence of the Cas12J protein, except where the sequence includes amino acid substitutions (e.g., 1, 2, or 3 amino acid substitutions) that reduce the catalytic activity of the protein in its native form. In some cases, the Cas12J polypeptide has a length of 750 to 790 amino acids (aa), such as 750 to 760 aa, 760 to 770 aa, 770 to 780 aa, or 780 to 790 aa. In some cases, the Cas12J polypeptide has a length of 772 amino acids. In some cases, the Cas12J polypeptide (e.g., containing amino acids described in...) is bound to... FIG. 6Q The guide RNA of the Cas12J polypeptide having 20% ​​or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% amino acid sequence identity includes the following nucleotide sequence: GTCTCAGCGT ACTGAGCAAT CAAAAGGTTTCGCAGG (SEQ ID NO: 13) or its reverse complement. In some cases, the guide RNA comprises the nucleotide sequence (N)nGTCTCAGCG TACTGAGCAA TCAAAAGGTT TCGCAGG (SEQ ID NO: 14) or its reverse complement, wherein N is any nucleotide and n is an integer from 15 to 30 (e.g., 15 to 20, 17 to 25, 17 to 22, 18 to 22, 18 to 20, 20 to 25, or 25 to 30).

[0147] In some cases, the Cas12J protein (of the compositions and / or methods of the present invention) comprises the protein described in FIG. 6Q The document specifies that the Cas12J amino acid sequence of “Cas12J_3877103_16” has 20% or higher sequence identity (e.g., 30% or higher, 40% or higher, 50% or higher, 60% or higher, 70% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity). For example, in some cases, the Cas12J protein includes amino acids that are described in [the document / reference needed]. FIG. 6QThe Cas12J amino acid sequence in the diagram has 50% or higher sequence identity (e.g., 60% or higher, 70% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity). In some cases, the Cas12J protein includes amino acids that are described in the diagram. FIG. 6Q The Cas12J amino acid sequence in the literature has 80% or higher sequence identity (e.g., 85% or higher, 90% or higher, 95% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity). In some cases, the Cas12J protein includes amino acids that are similar to those described in the literature. FIG. 6Q The Cas12J amino acid sequence has 90% or higher sequence identity (e.g., 95% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity). In some cases, the Cas12J protein includes amino acids depicted in... FIG. 6Q The amino acid sequence of the Cas12J protein. In some cases, the Cas12J protein includes amino acids depicted in [the original text]. FIG. 6Q The amino acid sequence of the Cas12J protein, except where the sequence includes amino acid substitutions (e.g., 1, 2, or 3 amino acid substitutions) that reduce the catalytic activity of the protein in its native form. In some cases, the Cas12J polypeptide has a length of 750 to 790 amino acids (aa), such as 750 to 760 aa, 760 to 770 aa, 770 to 780 aa, or 780 to 790 aa. In some cases, the Cas12J polypeptide has a length of 765 amino acids. In some cases, the Cas12J polypeptide (e.g., containing amino acids described in...) is bound to... FIG. 6RThe guide RNA of the Cas12J polypeptide having amino acid sequence identity of 20% or higher, 30% or higher, 40% or higher, 50% or higher, 60% or higher, 70% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% amino acid sequence identity includes the following nucleotide sequence: GTCGCGGCGT ACCGCGCAAT GAGAGTCTGTTGCCAT (SEQ ID NO: 21) or its reverse complement. In some cases, the guide RNA comprises the nucleotide sequence (N)nGTCGCGGCG TACCGCGCAA TGAGAGTCTG TTGCCAT (SEQ ID NO: 22) or its reverse complement, wherein N is any nucleotide and n is an integer from 15 to 30 (e.g., 15 to 20, 17 to 25, 17 to 22, 18 to 22, 18 to 20, 20 to 25, or 25 to 30).

[0148] In some cases, the Cas12J protein (of the compositions and / or methods of the present invention) comprises the protein described in FIG. 6R The document specifies that the Cas12J amino acid sequence of “Cas12J_877636_12” has 20% or higher sequence identity (e.g., 30% or higher, 40% or higher, 50% or higher, 60% or higher, 70% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity). For example, in some cases, the Cas12J protein includes amino acids that are described in [the document / reference]. FIG. 6R The Cas12J amino acid sequence in the diagram has 50% or higher sequence identity (e.g., 60% or higher, 70% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity). In some cases, the Cas12J protein includes amino acids that are described in the diagram. FIG. 6R The Cas12J amino acid sequence in the literature has 80% or higher sequence identity (e.g., 85% or higher, 90% or higher, 95% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity). In some cases, the Cas12J protein includes amino acids that are similar to those described in the literature. FIG. 6R The Cas12J amino acid sequence has 90% or higher sequence identity (e.g., 95% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity). In some cases, the Cas12J protein includes amino acids depicted in... FIG. 6RThe amino acid sequence of the Cas12J protein. In some cases, the Cas12J protein includes amino acids depicted in [the original text]. FIG. 6R The amino acid sequence of the Cas12J protein, except where the sequence includes amino acid substitutions (e.g., 1, 2, or 3 amino acid substitutions) that reduce the catalytic activity of the protein in its native form. In some cases, the Cas12J polypeptide has a length of 750 to 790 amino acids (aa), such as 750 to 760 aa, 760 to 770 aa, 770 to 780 aa, or 780 to 790 aa. In some cases, the Cas12J polypeptide has a length of 766 amino acids. In some cases, the Cas12J polypeptide (e.g., containing amino acids described in...) is bound to... FIG. 6A-6R The guide RNA of the Cas12J polypeptide having amino acid sequence identity of 20% or higher, 30% or higher, 40% or higher, 50% or higher, 60% or higher, 70% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% amino acid sequence identity includes the following nucleotide sequence: ACCAAAACGA CTATTGATTG CCCAGTACGCTGGGAC (SEQ ID NO: 23) or its reverse complement. In some cases, the guide RNA comprises the nucleotide sequence (N)nACCAAAACG ACTATTGATT GCCCAGTACG CTGGGAC (SEQ ID NO: 24) or its reverse complement, wherein N is any nucleotide and n is an integer from 15 to 30 (e.g., 15 to 20, 17 to 25, 17 to 22, 18 to 22, 18 to 20, 20 to 25, or 25 to 30).

[0149]

Cas12J variant

[0150] When compared with the amino acid sequence of the corresponding wild-type Cas12J protein, for example, when compared with the sequence described in... FIG. 6A-6R When compared to the Cas12J amino acid sequence in any of the variants, the variant Cas12J protein has at least one amino acid sequence difference (e.g., deletion, insertion, substitution, fusion). In some cases, compared to the Cas12J amino acid sequence described in [the original text], [the variant Cas12J protein has at least one different amino acid sequence (e.g., deletion, insertion, substitution, fusion).] FIG. 6A-6R Compared to the Cas12J amino acid sequence in any of them, the Cas12J variants contain 1 to 10 amino acid substitutions. In some cases, compared to the Cas12J amino acid sequence described in FIG. 9 Compared to the Cas12J amino acid sequence in any of them, the Cas12J variant contains 1 to 10 amino acid substitutions in the RuvC domain.

[0151] Variants - Catalytic Activity

[0152] In some cases, a Casl2J protein is a variant Casl2J protein, e.g., a protein that is mutated relative to a naturally occurring catalytically active sequence, and exhibits reduced cleavage activity when compared to the corresponding naturally occurring sequence (e.g., exhibits 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, or 30% or less cleavage activity). In some cases, such a variant Casl2J protein is a catalytically “dead” protein (substantially no cleavage activity) and can be referred to as a ‘dCasl2J’. In some cases, a variant Casl2J protein is a nickase (cleaves only one strand of a double stranded target nucleic acid, e.g., a double stranded target DNA). As described in greater detail herein, in some cases, a Casl2J protein (in some cases, a Casl2J protein with wild type cleavage activity and in some cases, a variant Casl2J with reduced cleavage activity, e.g., a dCasl2J or a nickase Casl2J) is fused (conjugated) to a heterologous polypeptide having an activity of interest (e.g., a catalytic activity of interest) to form a fusion protein (a fusion Casl2J protein).

[0153] Amino acid substitutions that yield Casl2J polypeptides that bind but do not cleave a target nucleic acid when complexed with a Casl2J guide RNA are depicted in FIG. 6D For example, substitution of an Asp at position 464 of Casl2J_l0037042_3 or at a corresponding position of another Casl2J yields a dCasl2J. As another example, substitution of a Glu at position 678 of Casl2J_l0037042_3 or at a corresponding position of another Casl2J yields a dCasl2J. As another example, substitution of an Asp at position 769 of Casl2J_l0037042_3 or at a corresponding position of another Casl2J yields a dCasl2J.

[0154] Amino acid substitutions that yield a dCasl2J polypeptide (i.e., a Casl2J polypeptide that binds but does not cleave a target nucleic acid when complexed with a guide RNA) include substitution of an Asp at position 413 of Casl2J_3339380( FIG. 6D ) or at a corresponding position of another Casl2J with an amino acid other than Asp. For example, amino acid substitutions that yield a dCasl2J polypeptide (i.e., a Casl2J polypeptide that binds but does not cleave a target nucleic acid when complexed with a guide RNA) include a D413A substitution at position 413 of Casl2J_3339380( FIG. 6A ) or at a corresponding position of another Casl2J.

[0155] The amino acid substitutions that produce the dCas12J polypeptide (i.e., the Cas12J polypeptide that binds to but does not cleave the target nucleic acid when complexed with guide RNA) include the substitution of non-Asp amino acids in Cas12J_1947455 ( FIG. 6A The Asp at position 371 of Cas12J or at the corresponding position in another Cas12J. For example, the amino acid substitutions that produce the dCas12J polypeptide (i.e., the Cas12J polypeptide that binds to but does not cleave the target nucleic acid when complexed with the guide RNA) include Cas12J_1947455 ( FIG. 6B Replace D371A at position 371 or at the corresponding position in another Cas12J.

[0156] The amino acid substitutions that produce the dCas12J polypeptide (i.e., the Cas12J polypeptide that binds to but does not cleave the target nucleic acid when complexed with guide RNA) include the substitution of non-Asp amino acids in Cas12J_2071242 ( FIG. 6B The Asp at position 394 of Cas12J or at the corresponding position in another Cas12J. For example, the amino acid substitutions that produce the dCas12J polypeptide (i.e., the Cas12J polypeptide that binds to but does not cleave the target nucleic acid when complexed with the guide RNA) include Cas12J_2071242 ( FIG. 6D Replace D394A at position 394 or at the corresponding position in another Cas12J.

[0157] Corresponding to Cas12J_3339380 ( FIG. 6A Asp at position 413 of (CasΦ-3), in Cas12J_1947455 ( FIG. 6B Asp at position 371 of (CasΦ-1) and at Cas12J_2071242 ( FIG. 6A-6R The amino acid position of Asp at position 394 of (CasΦ-2) can be easily determined by, for example, by comparing with the amino acid position of Asp. FIG. 6D The amino acid sequence of the Cas12J polypeptide is used to determine its composition. For example, it corresponds to Cas12J_3339380 ( FIG. 6A Asp at position 413, in Cas12J_1947455 ( FIG. 6B Asp at position 371 and at Cas12J_2071242 ( FIG. 9 The amino acid position of Asp at position 394 is depicted in FIG. 9 In, for example, the Asp that can produce the dCas12J polypeptide in Ruv-CI when substituted with non-Asp amino acids include:

[0158] (1) Specify “Cas12J_1947455” (or FIG. 6A(Cas12J_1947455_11) and depicted in FIG. 6B Asp-371 of the Cas12J polypeptide (“CasΦ-1”);

[0159] (2) Specify “Cas12J_2071242” and draw it in FIG. 9 Asp-394 of the Cas12J polypeptide (“CasΦ-2”);

[0160] (3) Specify “Cas12J_3339380” (or FIG. 6D ("Cas12J_3339380_12") and depicted in FIG. 6Q Asp-413 of the Cas12J polypeptide (“CasΦ-3”);

[0161] (4) Specify “Cas12J_3877103_16” and draw on FIG. 6G Asp-419 of the Cas12J polypeptide (“CasΦ-4”);

[0162] (5) Specify "Cas12J_10000002_47" or "Cas12J_1000002_112" and draw on FIG. 6H Asp-416 of the Cas12J polypeptide (“CasΦ-5”);

[0163] (6) Specify “Cas12J_10100763_4” and draw on FIG. 6P Asp-384 of the Cas12J polypeptide (“CasΦ-6”);

[0164] (7) Specify "Cas12J_1000007_143" or "Cas12J_1000001_267" and draw on FIG. 6L Asp-423 of the Cas12J polypeptide (“CasΦ-7”);

[0165] (8) Specify “Cas12J_10000286_53” and draw it in FIG. 6O (or “Cas12J_10000506_8” and described in) FIG. 6M Asp-369 of the Cas12J polypeptide (“CasΦ-8”) in the middle;

[0166] (9) Specify “Cas12J_10001283_7” and draw on FIG. 6E Asp-426 of the Cas12J polypeptide (“CasΦ-9”);

[0167] (10) Designated "Cas12J_10037042_3" and depicted in FIG. 10B. FIG. 13 Asp-464 of the Cas12J polypeptide ("CasΦ-10") in FIG. 10B.

[0168]

Variant - Fusion Cas12J Polypeptides

[0169] As described above, in some cases, a Cas12J protein (in some cases, a Cas12J protein with wild-type cleavage activity and in some cases, a variant Cas12J with reduced cleavage activity, e.g., a dCas12J or a nickase Cas12J) is fused (conjugated) to a heterologous polypeptide (i.e., one or more heterologous polypeptides) with an activity of interest (e.g., a catalytic activity of interest) to form a fusion protein. Heterologous polypeptides that can be fused to a Cas12J protein are referred to herein as "fusion partners."

[0170] In some cases, a fusion partner can modulate transcription of a target DNA (e.g., inhibit transcription, increase transcription). For example, in some cases, a fusion partner is a protein (or a domain from a protein) that inhibits transcription (e.g., a transcriptional repressor, which is a protein that acts via recruitment of transcriptional repressor proteins, modification of the target DNA (e.g., methylation), recruitment of DNA modification enzymes, modulation of histones associated with the target DNA, recruitment of histone modification enzymes (e.g., those that modify histones by acetylation and / or methylation), etc.). In some cases, a fusion partner is a protein (or a domain from a protein) that increases transcription (e.g., a transcriptional activator, which is a protein that acts via recruitment of transcriptional activator proteins, modification of the target DNA (e.g., demethylation), recruitment of DNA modification enzymes, modulation of histones associated with the target DNA, recruitment of histone modification enzymes (e.g., those that modify histones by acetylation and / or methylation), etc.). In some cases, a fusion partner is a reverse transcriptase. In some cases, a fusion partner is a base editor. In some cases, a fusion partner is a deaminase.

[0171] In some cases, a fusion Cas12J protein includes a heterologous polypeptide with an enzymatic activity that modifies a target nucleic acid (e.g., nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deaminase activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer formation activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity, or glycosylase activity).

[0172] In some cases, the fusion Cas12J protein includes a heterologous polypeptide having an enzymatic activity that modifies a polypeptide associated with the target nucleic acid (e.g., a histone) (e.g., a methyltransferase activity, a demethylase activity, an acetyltransferase activity, a deacetylase activity, a kinase activity, a phosphatase activity, a ubiquitin ligase activity, a deubiquitinating activity, an adenylation activity, a deadenylation activity, a SUMOylation activity, a desumoylation activity, a ribosylation activity, a deribosylation activity, a myristoylation activity, or a demyristoylation activity).

[0173] Examples of proteins (or fragments thereof) that can be used to increase transcription include, but are not limited to: transcriptional activators such as VP16, VP64, VP48, VP160, p65 subdomain (e.g., from NFkB), and activation domains of EDLL and / or TAL activation domains (e.g., for activity in plants); histone lysine methyltransferases such as SET1A, SET1B, MLL1-5, ASH1, SYMD2, NSD1, etc.; histone lysine demethylases such as JHDM2a / b, UTX, JMJD3, etc.; histone acetyltransferases such as GCN5, PCAF, CBP, p300, TAF1, TIP60 / PLIP, MOZ / MYST3, MORF / MYST4, SRC1, ACTR, P160, CLOCK, etc.; and DNA demethylases such as 10-11 translocation (TET) dioxygenase 1 (TET1CD), TET1, DME, DML1, DML2, ROS1, etc.

[0174] Examples of proteins (or fragments thereof) that can be used to reduce transcription include, but are not limited to: transcriptional repressors, such as Krüppel- associated box (KRAB or SKD); KOx1 repressor domain; Mad mSIN3 interaction domain (SID); ERF repressor domain (ERD), SRDX repressor domain (e.g., for repression in plants), and the like; histone lysine methyltransferases, such as Pr-SET7 / 8, SUV4-20H1, RIZ1, and the like; histone lysine demethylases, such as JMJD2A / JHDM3A, JMJD2B, JMJD2C / GASC1, JMJD2D, JARID1A / RBP2, JARID1B / PLU-1, JARID1C / SMCX, JARID1D / SMCY, and the like; histone lysine deacetylases, such as HDAC1, HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRT1, SIRT2, HDAC11, and the like; DNA methylases, such as Hhal DNA m5c-methyltransferase (M. Hhal), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3 (plant), ZMET2, CMT1, CMT2 (plant), and the like; and peripheral recruitment elements, such as lamin A, lamin B, and the like.

[0175] In some cases, the fusion partner has an enzymatic activity that modifies a target nucleic acid (e.g., ssRNA, dsRNA, ssDNA, dsDNA). Examples of enzymatic activities that can be provided by a fusion partner include, but are not limited to: nuclease activity, such as that provided by a restriction enzyme (e.g., Fokl nuclease); methyltransferase activity, such as that provided by a methyltransferase (e.g., Hhal DNA m5c-methyltransferase (M.Hhal), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), MET I, DRM3 (plant), ZMET2, CMT1, CMT2 (plant), etc.); demethylase activity, such as that provided by a demethylase (e.g.; 10-11 translocation (TET) dioxygenase 1 (TET1CD), TET1, DME, DML1, DML2, ROS1, etc.); DNA repair activity; DNA damage activity; deaminase activity, such as that provided by a deaminase (e.g., cytosine deaminase, such as rat APOBEC1); dismutase activity; alkylation activity; depurination activity; oxidation activity; pyrimidine dimer formation activity; integrase activity, such as that provided by an integrase and / or resolvase (e.g., Gin invertase such as the hyperactive mutant of Gin invertase Gin H106Y, human immunodeficiency virus type 1 integrase (IN), Tn3 resolvase, etc.); transposase activity; recombinase activity, such as that provided by a recombinase (e.g., catalytic domain of Gin recombinase); polymerase activity; ligase activity; helicase activity; photolyase activity, and glycosylase activity.

[0176] In some cases, the fusion partner has an enzymatic activity that modifies a protein (e.g., a histone, an RNA-binding protein, a DNA-binding protein, etc.) associated with a target nucleic acid (e.g., ssRNA, dsRNA, ssDNA, dsDNA). Examples of enzymatic activities (modify a protein associated with a target nucleic acid) that can be provided by a fusion partner include, but are not limited to: methyltransferase activity, such as that provided by a histone methyltransferase (HMT) (e.g., Suiable Inhibitor of Deformation 3-9 Homolog 1 (SUV39H1, also known as KMT1A), Euchromatic Histone-Lysine N-methyltransferase 2 (G9A, also known as KMT1C and EHMT2), SUV39H2, ESET / SETDB1, etc., SET1A, SET1B, MLL1-5, ASH1, SYMD2, NSD1, DOT1L, Pr-SET7 / 8, SUV4-20H1, EZH2, RIZ1); demethylase activity, such as that provided by a histone demethylase (e.g., lysine demethylase 1A (KDM1A, also known as LSD1), JHDM2a / b, JMJD2A / JHDM3A, JMJD2B, JMJD2C / GASC1, JMJD2D, JARID1A / RBP2, JARID1B / PLU-1, JARID1C / SMCX, JARID1D / SMCY, UTX, JMJD3, etc.); acetyltransferase activity, such as that provided by a histone acetyltransferase (e.g., catalytic core / fragment of human acetyltransferase p300, GCN5, PCAF, CBP, TAF1, TIP60 / PLIP, MOZ / MYST3, MORF / MYST4, HBO1 / MYST2, HMOF / MYST1, SRC1, ACTR, P160, CLOCK, etc.); deacetylase activity, such as that provided by a histone deacetylase (e.g., HDAC1, HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRT1, SIRT2, HDAC11, etc.); kinase activity; phosphatase activity; ubiquitin ligase activity; deubiquitinating activity; adenylation activity; deadenylation activity; SUMOylation activity; deSUMOylation activity; ribosylation activity; de-ribosylation activity; myristoylation activity and de-myristoylation activity.

[0177] Further examples of suitable fusion partners are dihydrofolate reductase (DHFR) destabilization domains (e.g., to generate a chemically controllable fusion Cas12J protein) and a chloroplast transit peptide. Suitable chloroplast transit peptides include, but are not limited to:

[0178] MASMISSSAV TTVSRASRGQ SAAMAPFGGL KSMTGFPVRK VNTDITSITS NGGRVKCMQVWPPIGKKKFE TLSYLPPLTR DSRA (SEQ ID NO: 25); MASMISSSAV TTVSRASRGQ SAAMAPFGGL KSMTGFPVRK VNTDITSITS NGGRVKS (SEQ ID NO: 26); MASSMLSSAT MVASPAQATM VAPFNGLKSSAAFPATRKAN NDITSITSNG GRVNCMQVWP PIEKKKFETL SYLPDLTDSG GRVNC (SEQ ID NO: 27); MAQVSRICNG VQNPSLISNL SKSSQRKSPL SVSLKTQQHP RAYPISSSWG LKKSGMTLIG SELRPLKVMSSVSTAC (SEQ ID NO: 28); MAQVSRICNG VWNPSLISNL SKSSQRKSPL SVSLKTQQHP RAYPISSSWGLKKSGMTLIG SELRPLKVMS SVSTAC (SEQ ID NO: 29); MAQINNMAQG IQTLNPNSNF HKPQVPKSSSFLVFGSKKLK NSANSMLVLK KDSIFMQLFC SFRISASVAT AC (SEQ ID NO: 30); MAALVTSQLATSGTVLSVTD RFRRPGFQGL RPRNPADAAL GMRTVGASAA PKQSRKPHRF DRRCLSMVV (SEQ ID NO: 31); MAALTTSQLAT SATGFGIAD RSAPSSLLRH GFQGLKPRSP AGGDATSLSV TTSARATPKQQRSVQRGSRR FPSVVVC (SEQ ID NO: 32); MASSVLSSAA VATRSNVAQA NMVAPFTGLK SAASFPVSRKQNLDITSIAS NGGRVQC (SEQ ID NO: 33); MESLAATSVF APSRVAVPAA RALVRAGTVV PTRRTSSTSGTSGVKCSAAV TPQASPVISR SAAAA (SEQ ID NO: 34);and MGAAATSMQS LKFSNRLVPPSRRLSPVPNN VTCNNLPKSA APVRTVKCCA SSWNSTINGA AATTNGASAA SS (SEQ ID NO: 35).

[0179] In some cases, a Casl2J fusion polypeptide of the present disclosure comprises: a) a Casl2J polypeptide of the present disclosure; and b) a chloroplast transit peptide. Thus, for example, a Casl2J polypeptide / guide RNA complex can be targeted to the chloroplast. In some cases, such targeting can be achieved by the presence of an N-terminal extension known as a chloroplast transit peptide (CTP) or plastid transit peptide. If a polypeptide expressed is to be compartmentalized in the plant plastid (e.g., chloroplast), the chromosomal transgene from a bacterial source must have a sequence encoding a CTP sequence fused to the sequence encoding the expressed polypeptide. Thus, localization of a foreign polypeptide to the chloroplast is typically achieved by operably linking a polynucleotide sequence encoding a CTP sequence to the 5’ region of a polynucleotide encoding a foreign polypeptide. During translocation into the plastid, the CTP is removed in a processing step. However, processing efficiency can be affected by the amino acid sequence of the CTP and the sequence near the amino-terminal (NH2-terminal) end of the peptide. Other options for targeting to the chloroplast that have been described are the maize cab-m7 signal sequence (U.S. Patent No. 7,022,896, WO 97 / 41228), the pea glutathione reductase signal sequence (WO 97 / 41228), and the CTP described in US2009029861.

[0180] In some cases, a Casl2J fusion polypeptide of the present disclosure can comprise: a) a Casl2J polypeptide of the present disclosure; and b) an endosomal escape peptide. In some cases, the endosomal escape polypeptide comprises the amino acid sequence GLFXALLXLL XSLWXLLLXA (SEQ ID NO: 36), where each X is independently selected from lysine, histidine, and arginine. In some cases, the endosomal escape polypeptide comprises the amino acid sequence GLFHALLHLL HSLWHLLLHA (SEQ ID NO: 37).

[0181] For examples of some of the above fusion partners (and more) used in fusion with Cas9 proteins, zinc finger proteins, and / or TALE proteins (for site-specific target nucleic acid modification, transcriptional modulation, and / or target protein modification, e.g., histone modification), see, e.g.: Nomura et al., J Am Chem Soc. 2007 Jul 18;129(28):8676-7; Rivenbark et al., Epigenetics. 2012 Apr;7(4):350-60; Nucleic Acids Res. 2016 Jul 8;44(12):5615-28; Gilbert et al., Cell. 2013 Jul 18;154(2):442-51; Kearns et al., Nat Methods. 2015 May;12(5):401-3; Mendenhall et al., Nat Biotechnol. 2013 Dec;31(12):1133-6; Hilton et al., Nat Biotechnol. 2015 May;33(5):510-7; Gordley et al., Proc Natl Acad Sci U S A. 2009 Mar 31;106(13):5053-8; Akopian et al., Proc Natl Acad Sci U S A. 2003 Jul 22;100(15):8688-91; Tan et al., J Virol. 2006 Feb;80(4):1939-48; Tan et al., Proc Natl Acad Sci U S A. 2003 Oct 14;100(21):11997-2002; Papworth et al., Proc Natl Acad Sci U S A. 2003 Feb 18;100(4):1621-6; Sanjana et al., Nat Protoc. 2012 Jan 5;7(1):171-92; Beerli et al., Proc Natl Acad Sci U S A. 1998 Dec 8;95(25):14628-33; Snowden et al., Curr Biol. 2002 Dec 23;12(24):2159-66; Xu et al., Cell Discov. 2016 May 3;2:16009; Komor et al., Nature. 2016 Apr 20;533(7603):420-4; Chaikind et al., Nucleic Acids Res. 2016 Aug 11; Choudhury et al., Oncotarget.June 23, 2016; Du et al., Cold Spring Harb Protoc. 2016 Jan 4; Pham et al., Methods Mol Biol. 2016; 1358:43-57; Balboa et al., Stem Cell Reports. 2015 Sep 8; 5(3):448-59; Hara et al., Sci Rep. 2015 Jun 9; 5:11221; Piatek et al., Plant Biotechnol J. 2015 May; 13(4):578-89; Hu et al., Nucleic Acids Res. 2014 Apr; 42(7):4375-90; Cheng et al., Cell Res. 2013 Oct; 23(10):1163-71; and Maeder et al., Nat Methods. 2013 Oct; 10(10):977-9.

[0182] Further suitable heterologous polypeptides include, but are not limited to, polypeptides that directly and / or indirectly provide for increased or decreased transcription and / or translation of a target nucleic acid (e.g., transcriptional activators or fragments thereof, proteins or fragments thereof that recruit transcriptional activators, small molecule / drug responsive transcriptional and / or translational regulators, translational regulatory proteins, etc.). Non-limiting examples of heterologous polypeptides that effect increased or decreased transcription include transcriptional activator domains and transcriptional repressor domains. In some such cases, a fusion Cas12J polypeptide is targeted by a guide nucleic acid (guide RNA) to a particular location (i.e., sequence) in a target nucleic acid and functions to exert locus-specific regulation, such as blocking the binding of RNA polymerase to a promoter that selectively inhibits transcriptional activator function and / or modifying local chromatin state (e.g., when using a fusion sequence that modifies a target nucleic acid or modifies a polypeptide associated with a target nucleic acid (e.g., a nucleosome histone)). In some cases, the change is temporary (e.g., transcriptional repression or activation). In some cases, the change is heritable (e.g., when epigenetic modification of a target nucleic acid or a protein associated with a target nucleic acid (e.g., a nucleosome histone) is made).

[0183] Non-limiting examples of heterologous polypeptides used when targeting ssRNA target nucleic acids include, but are not limited to: splicing factors (e.g., RS domains); protein translation components (e.g., translation initiation factors, elongation factors, and / or release factors; e.g., eIF4G); RNA methylases; RNA editing enzymes (e.g., RNA deaminases, such as adenosine deaminases that act on RNA (ADARs), including A-to-I and / or C-to-U editing enzymes); helicases; RNA binding proteins, etc. It will be appreciated that a heterologous polypeptide can include an entire protein, or in some cases, can include a fragment (e.g., a functional domain) of a protein.

[0184] A heterologous polypeptide of a subject fusion Casl2J polypeptide can be any domain capable of interacting with ssRNA (including intramolecular and / or intermolecular secondary structure for purposes of the present disclosure, e.g., double-stranded RNA duplexes such as hairpins, stem loops, etc.), whether transient or irreversible, direct or indirect, including but not limited to an effector domain selected from the group consisting of: endonucleases (e.g., RNase III, CRR22 DYW domain, Dicer from proteins such as SMG5 and SMG6, and PIN (PilT N-terminal) domain); proteins and protein domains responsible for stimulating RNA cleavage (e.g., CPSF, CstF, CFIm, and CFIIm); exonucleases (e.g., XRN-1 or Exonuclease T); de-adenylases (e.g., HNT3); proteins and protein domains responsible for nonsense-mediated RNA decay (e.g., UPF1, UPF2, UPF3, UPF3b, RNP S1, Y14, DEK, REF2, and SRm160); proteins and protein domains responsible for stabilizing RNA (e.g., PABP); proteins and protein domains responsible for repressing translation (e.g., Ago2 and Ago4); proteins and protein domains responsible for stimulating translation (e.g., Staufen); proteins and protein domains responsible for (e.g., capable of) modulating translation (e.g., translation factors such as initiation factors, elongation factors, release factors, etc., e.g., eIF4G); proteins and protein domains responsible for polyadenylation of RNA (e.g., PAP1, GLD-2, and Star-PAP); proteins and protein domains responsible for polyuridylation of RNA (e.g., CID1 and terminal uridylyltransferase); proteins and protein domains responsible for RNA localization (e.g., from IMP1, ZBP1, She2p, She3p, and Bicaudal-D); proteins and protein domains responsible for nuclear retention of RNA (e.g., Rrp6); proteins and protein domains responsible for nuclear export of RNA (e.g., TAP, NXF1, THO, TREX, REF, and Aly); proteins and protein domains responsible for repressing RNA splicing (e.g., PTB, Sam68, and hnRNP A1); proteins and protein domains responsible for stimulating RNA splicing (e.g., serine / arginine (SR) domains); proteins and protein domains responsible for decreasing transcriptional efficiency (e.g., FUS (TLS)); and proteins and protein domains responsible for stimulating transcription (e.g., CDK7 and HIV Tat).Alternatively, the effector domain can be selected from the group comprising endonucleases; proteins and protein domains capable of stimulating RNA cleavage; exonucleases; deaminases; proteins and protein domains with nonsense-mediated RNA decay activity; proteins and protein domains capable of stabilizing RNA; proteins and protein domains capable of repressing translation; proteins and protein domains capable of stimulating translation; proteins and protein domains capable of modulating translation (e.g., translation factors such as initiation factors, elongation factors, release factors, etc., e.g., eIF4G); proteins and protein domains capable of performing polyadenylation of RNA; proteins and protein domains capable of performing polyuridylation of RNA; proteins and protein domains with RNA localization activity; proteins and protein domains capable of performing nuclear retention of RNA; proteins and protein domains with RNA nuclear export activity; proteins and protein domains capable of repressing RNA splicing; proteins and protein domains capable of stimulating RNA splicing; proteins and protein domains capable of decreasing transcription efficiency; and proteins and protein domains capable of stimulating transcription. Another suitable heterologous polypeptide is the PUFRNA binding domain, which is described in more detail in WO2012068627, which is incorporated by reference herein in its entirety.

[0185] Some RNA splicing factors, which can be used as heterologous peptides (whole or as fragments) fused to Cas12J peptides, have modular structures with separate sequence-specific RNA-binding modules and splicing effector domains. For example, members of the serine / arginine-rich (SR) protein family contain an N-terminal RNA recognition motif (RRM) that binds to the exon splicing enhancer (ESE) in the pre-mRNA and C-terminal RS domain, which promotes exon inclusion. As another example, the hnRNP protein hnRNP A1 binds to an exon splicing silencer (ESS) via its RRM domain and inhibits exon inclusion via its C-terminal glycine-rich domain. Some splicing factors regulate the alternative use of splice sites (SSs) by binding to regulatory sequences between two alternative sites. For example, ASF / SF2 recognizes an ESE and facilitates the use of intron-proximal sites, while hnRNP A1 binds to an ESS and redirects splicing to the use of intron-distal sites. One application of such factors is the generation of alternative splicing factors (ESFs) that regulate the splicing of endogenous genes, particularly disease-related genes. For example, Bcl-x premRNA produces two splice isoforms with two alternative 5' splice sites encoding proteins with opposite functions. The long splice isoform, Bcl-xL, is a potent apoptosis inhibitor expressed in long-lived postmitotic cells and is upregulated in many cancer cells, thus protecting cells from apoptotic signals. The short isoform, Bcl-xS, is a pro-apoptotic isoform and is expressed at high levels in cells with high turnover rates, such as developing lymphocytes. The ratio of the two Bcl-x splice isoforms is determined by multiple [specific factors] located in the core exon region or exon extension region (i.e., between the two alternative 5' splice sites). Component control. For more examples, see WO2010075303, which is hereby incorporated in its entirety by reference.

[0186] Other suitable fusion partners include, but are not limited to, proteins (or fragments thereof) that serve as boundary elements (e.g., CTCF), proteins and fragments thereof that provide peripheral recruitment (e.g., lamin A, lamin B, etc.), and protein docking elements (e.g., FKBP / FRB, Pil1 / Aby1, etc.).

[0187] Nuclease

[0188] In some cases, a subject fusion Casl2J polypeptide comprises: i) a Casl2J polypeptide of the disclosure; and ii) a heterologous polypeptide (“fusion partner”), wherein the heterologous polypeptide is a nuclease. Suitable nucleases include, but are not limited to, a homing nuclease polypeptide; a Fokl polypeptide; a transcription activator-like effector nuclease (TALEN) polypeptide; a MegaTAL polypeptide; a meganuclease polypeptide; a zinc finger nuclease (ZFN); an ARCUS nuclease; and the like. The meganuclease can be engineered from a LADLIDADG homing endonuclease (LHE). A megaTAL polypeptide can comprise a TALE DNA binding domain and an engineered meganuclease. See, e.g., WO 2004 / 067736 (homing endonucleases); Urnov et al. (2005) Nature 435:646 (ZFN); Mussolino et al. (2011) Nucle. Acids Res. 39:9283 (TALE nucleases); Boissel et al. (2013) Nucl. Acids Res. 42:2591 (MegaTAL).

[0189]

Reverse Transcriptase

[0190] In some cases, a subject fusion Casl2J polypeptide comprises: i) a Casl2J polypeptide of the disclosure; and ii) a heterologous polypeptide (“fusion partner”), wherein the heterologous polypeptide is a reverse transcriptase polypeptide. In some cases, the Casl2J polypeptide is catalytically inactive. Suitable reverse transcriptases include, e.g., murine leukemia virus reverse transcriptase; Rous sarcoma virus reverse transcriptase; human immunodeficiency virus type I reverse transcriptase; Moloney murine leukemia virus reverse transcriptase; and the like.

[0191]

Base Editor

[0192] In some cases, a Casl2J fusion polypeptide of the disclosure comprises: i) a Casl2J polypeptide of the disclosure; and ii) a heterologous polypeptide (“fusion partner”), wherein the heterologous polypeptide is a base editor. Suitable base editors include, e.g., an adenosine deaminase; a cytidine deaminase (e.g., activation-induced cytidine deaminase (AID)); APOBEC3G; and the like); and the like.

[0193] A suitable adenosine deaminase is any enzyme capable of deaminating adenosine in DNA. In some cases, the deaminase is a TadA deaminase.

[0194] In some cases, a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: MSEVEFSHEY WM RHALTLAKR AWDEREVPVG AVLVHNNRVIGEGWNRPI GRHDPTAHAEI MALRQGGLVM QNYRLIDATL YVTLEPCVMC AGAMIHSRI GRVVFGARDAK TGAAGSLMDV LHHPGMNHRV EITEGILADE CAALLSDFFR MRRQEIKAQK KAQSSTD (SEQ ID NO: 38)

[0195] In some cases, a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: MRRAFITGVF FLSEVEFSHE YWM RHALTLAKR AWDEREVPVG AVLVHNNRVIGEGWNRPI GRHDPTAHAEI MALRQGGLVM QNYRLIDATL YVTLEPCVMC AGAMIHSRI GRVVFGARDAK TGAAGSLMDV LHHPGMNHRV EITEGILADE CAALLSDFFR MRRQEIKAQK KAQSSTD (SEQ ID NO: 39).

[0196] In some cases, a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following Staphylococcus aureus TadA amino acid sequence: MGSHMTNDIY FMTLAIEEAK KAAQLGEVPI GAIITKDDEV IARAHNLRETLQQPTAHAEH IAIERAAKVL GSWRLEGCTL YVTLEPCVMC AGTIVMSRIP RVVYGADDPK GGCSGSLMNLLQQSNFNHRA IVDKGVLKEA CSTLLTTFFK NLRANKKSTN: (SEQ ID NO: 40)

[0197] In some cases, a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following Bacillus subtilis TadA amino acid sequence: MTQDELYMKE AIKEAKKAEE KGEVPIGAVL VINGEIIARA HNLRETEQRSIAHAEMLVID EACKALGTWR LEGATLYVTL EPCPMCAGAV VLSRVEKVVF GAFDPKGGCS GTLMNLLQEERFNHQAEVVS GVLEEECGGM LSAFFRELRK KKKAARKNLS E (SEQ ID NO: 41)

[0198] In some cases, a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following Salmonella typhimurium TadA: MPPAFITGVT SLSDVELDHE YWMRHALTLA KRAWDEREVP VGAVLVHNHR VIGEGWNRPI GRHDPTAHAE I MALRQGGLV LQNYRLLDTT LYVTLEPCVM CAGAMVHSRI GRVVFGARDA KTGAAGSLID VLHHPGMNHR VEIIEGVLRD ECATLLSDFF RMRRQEIKAL KKADRAEGAG PAV (SEQ ID NO: 42)

[0199] In some cases, a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following Shewanella putrefaciens TadA amino acid sequence: MDEYWMQVAM QMAEKAEAAG EVPVGAVLVK DGQQIATGYN LSISQHDPTA HAEILCLRSA GKKLENYRLL DATLYITLEP CAMCAGAMVH SRIARVVYGA RDEKTGAAGT VVNLLQHPAF NHQVEVTSGV LAEACSAQLS RFFKRRRDEK KALKLAQRAQ QGIE (SEQ ID NO: 43)

[0200] In some cases, a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following H. influenzae F3031 TadA amino acid sequence: MDAAKVRSEFDEKMMRYALELADKAEALGEIPVGAVLVDDARNIIGEGWNLSIVQSDPTAHAEIIALRNGAKNIQNYRLLNSTLYVTLEPC TMCA GAILHSRIKRLVFGASD YKTGAIGSRFHFFDDYKMNHTLEITSGVLAEECSQKLS TFFQKRREEKKIEKALLKSLSDK (SEQ ID NO: 44)

[0201] In some cases, a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following C. crescentus TadA amino acid sequence: MRTDESEDQDHRMMRLALDAA RAAAEAGET PVGAVILDPS TGEVIATAGN GPIAAHDPTAHAEIAAMRAA AAKLGNYRLT DLTLVVTLEP CAMCAGAISH ARIGRVVFGA DDPKGGAVVH GPKFFAQPTCHWRPEVTGGV LADESADLLR GFFRARRKAK I (SEQ ID NO: 45)

[0202] In some cases, a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following S. reeganii TadA amino acid sequence: MSSLKKTPIRDDAYWMGKAI REAAKAAARD EVPIGAVIVR DGAVIGRGHNLREGSNDPSA HAEMIAIRQA ARRSANWRLT GATLYVTLEP CLMCMGAIIL ARLERVVFGC YDPKGGAAGSLYDLSADPRL NHQVRLSPGV CQEECGTMLSDFFRDLRRRK KAKATPALFIDERKVPPEP (SEQ ID NO: 46)

[0203] Cytidine deaminases suitable for inclusion in a CRISPR / Cas effector polypeptide fusion polypeptide include any enzyme capable of deaminating cytidine in DNA.

[0204] In some cases, the cytidine deaminase is a deaminase from the apolipoprotein B mRNA-editing complex (APOBEC) family of deaminases. In some cases, the APOBEC family deaminase is selected from the group consisting of APOBEC1 deaminase, APOBEC2 deaminase, APOBEC3A deaminase, APOBEC3B deaminase, APOBEC3C deaminase, APOBEC3D deaminase, APOBEC3F deaminase, APOBEC3G deaminase, and APOBEC3H deaminase. In some cases, the cytidine deaminase is activation-induced deaminase (AID).

[0205] In some cases, a suitable cytidine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence:

[0206] MDSLLMNRRK FLYQFKNVRW AKGRRETYLC YVVKRRDSAT SFSLDFGYLR NKNGCHVELL

[0207] FLRYISDWDL DPGRCYRVTW FTSWSPCYDC ARHVADFLRG NPNLSLRIFT ARLYFCEDRK

[0208] AEPEGLRRLH RAGVQIAIMT FKDYFYCWNT FVENHERTFK AWEGLHENSVRLSRQLRRILLPLYEVDDLR DAFRTLGL (SEQ ID NO: 47)

[0209] In some cases, a suitable cytidine deaminase is AID and comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: mdsllmnrrk flyqfknvrw akgrretylc yvvkrrdsat sfsldfgylr nkngchvell flryisdwdl dpgrcyrvtw ftswspcydc arhvadflrg npnlslrift arlyfcedrk aepeglrrlhragvqiaimt fkenhertfk aweglhensv rlsrqlrril lplyevddlr dafrtlgl (SEQ ID NO: 48).

[0210] In some cases, a suitable cytidine deaminase is AID and comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: mdsllmnrrk flyqfknvrw akgrretylc yvvkrrdsat sfsldfgylr nkngchvell flryisdwdl dpgrcyrvtw ftswspcydc arhvadflrg npnlslrift arlyfcedrk aepeglrrlhragvqiaimt fkdyfycwnt fvenhertfk aweglhensv rlsrqlrril lplyevddlr dafrtlgl (SEQ ID NO: 47).

[0211] [Transcription factor]

[0212] In some cases, a Cas12J fusion polypeptide of the disclosure comprises: i) a Cas12J polypeptide of the disclosure; and ii) a heterologous polypeptide (“fusion partner”), wherein the heterologous polypeptide is a transcription factor. A transcription factor can comprise: i) a DNA binding domain; and ii) a transcriptional activator. A transcription factor can comprise: i) a DNA binding domain; and ii) a transcriptional repressor. Suitable transcription factors include polypeptides having a transcriptional activator or transcriptional repressor domain (e.g., Kruppel-associated box (KRAB or SKD); Mad m SIN3 interaction domain (SID); ERF repressor domain (ERD), etc.); zinc-finger-based artificial transcription factors (see, e.g., Sera (2009) Adv. Drug Deliv. 61:513); TALE-based artificial transcription factors (see, e.g., Liu et al. (2013) Nat. Rev. Genetics 14:781); etc. In some cases, the transcription factor comprises a VP64 polypeptide (transcriptional activation). In some cases, the transcription factor comprises a Krüppel-associated box (KRAB) polypeptide (transcriptional repression). In some cases, the transcription factor comprises a Mad m SIN3 interaction domain (SID) polypeptide (transcriptional repression). In some cases, the transcription factor comprises an ERF repressor domain (ERD) polypeptide (transcriptional repression). For example, in some cases, the transcription factor is a transcriptional activator, wherein the transcriptional activator is GAL4-VP16.

[0213]

Recombinase

[0214] In some cases, a Cas12J fusion polypeptide of the disclosure comprises: i) a Cas12J polypeptide of the disclosure; and ii) a heterologous polypeptide (“fusion partner”), wherein the heterologous polypeptide is a recombinase. Suitable recombinases include, e.g., Cre recombinase; Hin recombinase; Tre recombinase; FLP recombinase; etc.

[0215] Examples of various additional suitable heterologous polypeptides (or fragments thereof) for fusion to a subject Casl2J polypeptide include, but are not limited to, those described in the following applications (the publications relate to other CRISPR endonucleases such as Cas9, but the fusion partners described can also be used with Casl2J): PCT Patent Application: WO2010075303, WO2012068627, and WO2013155555, and can be found, for example, in U.S. Patents and Patent Applications: 8,906,616; 8,895,308; 8,889,418; 8,889,356; 8,871,445; 8,865,406; 8,795,965; 8,771,945; 8,697,359; 20140068797; 20140170753; 20140179006; 20140179770; 20140186843; 20140186919; 20140186958; 20140189896; 20140227787; 20140234972; 20140242664; 20140242699; 20140242700; 20140242702; 20140248702; 20140256046; 20140273037; 20140273226; 20140273230; 20140273231; 20140273232; 20140273233; 20140273234; 20140273235; 20140287938; 20140295556; 20140295557; 20140298547; 20140304853; 20140309487; 20140310828; 20140310830; 20140315985; 20140335063; 20140335620; 20140342456; 20140342457; 20140342458; 20140349400; 20140349405; 20140356867; 20140356956; 20140356958; 20140356959; 20140357523; 20140357530; 20140364333; and 20140377868; which patents are all hereby incorporated by reference in their entirety.

[0216] In some cases, a heterologous polypeptide (fusion partner) provides subcellular localization, i.e., the heterologous polypeptide contains a subcellular localization sequence (e.g., a nuclear localization signal (NLS) for targeting the nucleus, a sequence for retaining the fusion protein outside the nucleus (e.g., a nuclear export sequence (NES)), a sequence for retaining the fusion protein in the cytoplasm, a mitochondrial localization signal for targeting mitochondria, a chloroplast localization signal for targeting chloroplasts, an ER retention signal, etc.). In some cases, a Casl2J fusion polypeptide does not include an NLS, such that the protein does not target the nucleus (which can be advantageous, e.g., when the target nucleic acid is an RNA that is present in the cytosol). In some cases, the heterologous polypeptide can provide a tag that facilitates tracking and / or purification (i.e., the heterologous polypeptide is a detectable marker) (e.g., a fluorescent protein, e.g., green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), mCherry, tdTomato, etc.; a histidine tag, e.g., a 6xHis tag; a hemagglutinin (HA) tag; a FLAG tag; a Myc tag; etc.).

[0217] In some cases, a Casl2J protein (e.g., a wild-type Casl2J protein, a variant Casl2J protein, a fusion Casl2J protein, a dCasl2J protein, etc.) includes (is fused to) a nuclear localization signal (NLS) (e.g., in some cases, 2 or more, 3 or more, 4 or more, or 5 or more NLS). Thus, in some cases, a Casl2J polypeptide comprises one or more NLS (e.g., 2 or more, 3 or more, 4 or more, or 5 or more NLS). In some cases, the one or more NLS (2 or more, 3 or more, 4 or more, or 5 or more NLS) are positioned at or near (e.g., within 50 amino acids of) the N-terminus and / or the C-terminus. In some cases, the one or more NLS (2 or more, 3 or more, 4 or more, or 5 or more NLS) are positioned at or near (e.g., within 50 amino acids of) the N-terminus. In some cases, the one or more NLS (2 or more, 3 or more, 4 or more, or 5 or more NLS) are positioned at or near (e.g., within 50 amino acids of) the C-terminus. In some cases, the one or more NLS (3 or more, 4 or more, or 5 or more NLS) are positioned at or near (e.g., within 50 amino acids of) both the N-terminus and the C-terminus. In some cases, an NLS is positioned at the N-terminus and an NLS is positioned at the C-terminus.

[0218] In some cases, a Cas12J protein (e.g., a wild type Cas12J protein, a variant Cas12J protein, a fusion Cas12J protein, a dCas12J protein, etc.) comprises (is fused to) between 1 and 10 NLSs (e.g., 1-9, 1-8, 1-7, 1-6, 1-5, 2-10, 2-9, 2-8, 2-7, 2-6, or 2-5 NLSs). In some cases, a Cas12J protein (e.g., a wild type Cas12J protein, a variant Cas12J protein, a fusion Cas12J protein, a dCas12J protein, etc.) comprises (is fused to) between 2 and 5 NLSs (e.g., 2-4 or 2-3 NLSs).

[0219] Non-limiting examples of NLSs include NLS sequences derived from the NLS of the SV40 virus large T antigen having the amino acid sequence PKKKRKV (SEQ ID NO: 49); from the NLS from nucleoplasmin (e.g., the bipartite NLS of nucleoplasmin having the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 50)); the c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 51) or RQRRNELKRS P (SEQ ID NO: 52); the hRNPA1 M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSG PYGGGGQYFA KPRNQGGY (SEQ ID NO: 53); the sequence RMRIZFKNKG KDTAELRRRR VEVSVELRKA KKDEQILKRR NV (SEQ ID NO: 54) from the IBB domain of importin-alpha; the sequences VSRKRPRP (SEQ ID NO: 55) and PPKKARED (SEQ ID NO: 98) of the myoma T protein; the sequence PQPKKKPL (SEQ ID NO: 56) of human p53; the sequence SALIKKKKKM AP (SEQ ID NO: 57) of mouse c-abl IV; the sequences DRLRR (SEQ ID NO: 58) and PKQKKRK (SEQ ID NO: 59) of influenza virus NS1; the sequence RKLKKKIKKL (SEQ ID NO: 60) of hepatitis delta antigen; the sequence REKKKFLKRR (SEQ ID NO: 61) of mouse Mxl protein; the sequence KRKGDEVDGV DEVAKKKSKK (SEQ ID NO: 62) of human poly(ADP-ribose) polymerase; and the sequence RKCLQAGMNL EARKTKK (SEQ ID NO: 63) of the steroid hormone receptor (human) glucocorticoid. In general, the NLS (or NLSs) is of sufficient strength to drive the Casl2J protein to accumulate in a detectable amount in the nucleus of a eukaryotic cell. Detection of accumulation in the nucleus can be performed by any suitable technique. For example, a detectable marker can be fused to the Casl2J protein, such that the location within the cell can be visualized. The nucleus can also be isolated from the cell, and the contents of the nucleus can then be analyzed by any suitable method for detecting proteins, such as immunohistochemistry, Western blot, or enzyme activity assays. Accumulation in the nucleus can also be determined indirectly.

[0220] In some cases, a Cas12J fusion polypeptide includes a “protein transduction domain” or PTD (also known as a CPP - cell-penetrating peptide), which refers to a polypeptide, polynucleotide, carbohydrate, or organic or inorganic compound that facilitates crossing of a lipid bilayer, micelle, cell membrane, organelle membrane, or vesicle membrane. A PTD attached to another molecule (which can range from a small polar molecule to a large macromolecule and / or nanoparticle) facilitates the molecule crossing a membrane, e.g., from an extracellular space into an intracellular space or from a cytosol into an organelle. In some embodiments, a PTD is covalently linked to the amino-terminal end of a polypeptide (e.g., linked to a wild-type Cas12J to generate a fusion protein, or linked to a variant Cas12J protein such as a dCas12J, a nickase Cas12J, or a fusion Cas12J protein to generate a fusion protein). In some embodiments, a PTD is covalently linked to the carboxy-terminal end of a polypeptide (e.g., linked to a wild-type Cas12J to generate a fusion protein, or linked to a variant Cas12J protein such as a dCas12J, a nickase Cas12J, or a fusion Cas12J protein to generate a fusion protein). In some cases, the PTD is intercalated in the Cas12J fusion polypeptide at a suitable insertion site (i.e., not at the N- or C-terminus of the Cas12J fusion polypeptide). In some cases, a subject Cas12J fusion polypeptide includes (is conjugated to, is fused to) one or more PTDs (e.g., two or more, three or more, four or more PTDs). In some cases, a PTD includes a nuclear localization signal (NLS) (e.g., in some cases, 2 or more, 3 or more, 4 or more, or 5 or more NLSs). Thus, in some cases, a Cas12J fusion polypeptide includes one or more NLSs (e.g., 2 or more, 3 or more, 4 or more, or 5 or more NLSs). In some embodiments, a PTD is covalently linked to a nucleic acid (e.g., a Cas12J guide nucleic acid, a polynucleotide encoding a Cas12J guide nucleic acid, a polynucleotide encoding a Cas12J fusion polypeptide, a donor polynucleotide, etc.).Examples of PTDs include, but are not limited to, the minimal eleven-amino acid polypeptide protein transduction domain (corresponding to residues 47-57 of HIV-1 TAT comprising YGRKKRRQRRR; SEQ ID NO:64); a polyarginine sequence comprising a number of arginines sufficient to directly enter a cell (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10-50 arginines); a VP22 domain (Zender et al. (2002) Cancer Gene Ther. 9(6):489-96); a Drosophila Antennapedia gene protein transduction domain (Noguchi et al. (2003) Diabetes 52(7): 1732-1737); a truncated human calcitonin peptide (Trehin et al. (2004) Pharm. Research 21 : 1248-1256); a polylysine (Wender et al. (2000) Proc. Natl. Acad. Sci. USA 97: 13003-13008); RRQRRTSKLM KR (SEQ ID NO:65); a transportan protein GWTLNSAGYL LGKINLKALAALAKKIL (SEQ ID NO:66); KALAWEAKLA KALAKALAKH LAKALAKALK CEA (SEQ ID NO:67); and RQIKIWFQNR RMKWKK (SEQ ID NO:68). Exemplary PTDs include, but are not limited to: YGRKKRRQRRR (SEQ ID NO:64), RKKRRQRRR (SEQ ID NO:70); an arginine homopolymer having 3 arginine residues to 50 arginine residues; exemplary PTD domain amino acid sequences include, but are not limited to, any one of: YGRKKRRQRRR (SEQ ID NO:64); RKKRRQRR (SEQ ID NO:70); YARAAARQARA (SEQ ID NO:71); THRLPRRRRRR (SEQ ID NO:72); and GGRRARRRRRR (SEQ ID NO:73). In some embodiments, the PTD is an activatable CPP (ACPP) (Aguilera et al. (2009) Integr Biol (Camb) Jun; 1(5-6):371-381). ACPPs include a polycationic CPP (e.g., Arg9 or "R9") linked via a cleavable linker to a matching polyanion (e.g., Glu9 or "E9"), which reduces the net charge to near zero and thereby inhibits adhesion and uptake into cells. When the linker is cleaved, the polyanion is released, locally exposing the polyarginine and its inherent adhesiveness, thereby "activating" the ACPP to cross the membrane.

[0221] [Linkers (e.g., for fusion partners)]

[0222] In some embodiments, a subject Cas12J protein can be fused to a fusion partner via a linker polypeptide (e.g., one or more linker polypeptides). The linker polypeptide can have any of a variety of amino acid sequences. Proteins can be linked by a spacer peptide, which typically has flexible properties, although other chemical linkages are not excluded. Suitable linkers include polypeptides between 4 and 40 amino acids in length or between 4 and 25 amino acids in length. These linkers can be produced by using synthetic oligonucleotides encoding the linker to couple the proteins, or can be encoded by the nucleic acid sequence encoding the fusion protein. Peptide linkers with some degree of flexibility can be used. The linking peptide can in fact have any amino acid sequence, with the understanding that preferred linkers will have sequences that result in a generally flexible peptide. The use of small amino acids, such as glycine and alanine, are useful in producing flexible peptides. Producing such sequences is routine to one of skill in the art. A variety of different linkers are commercially available and are considered suitable for use.

[0223] Examples of linker polypeptides include glycine polymers (G) n , glycine-serine polymers (including, e.g., (GS) n , GSGGS n (SEQ ID NO: 74), GGSGGS n (SEQ ID NO: 75), and GGGS n (SEQ ID NO: 76), where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers. Exemplary linkers can comprise amino acid sequences including, but not limited to, GGSG (SEQ ID NO: 77), GGSGG (SEQ ID NO: 78), GSGSG (SEQ ID NO: 79), GSGGG (SEQ ID NO: 80), GGGSG (SEQ ID NO: 81), GSSSG (SEQ ID NO: 82), and the like. One of ordinary skill will recognize that the design of a peptide conjugated to any desired element can include a linker that is wholly or partially flexible, such that the linker can include a flexible linker as well as one or more portions that impart a less flexible structure.

[0224] [Detectable label]

[0225] In some cases, a Cas12J polypeptide of the disclosure comprises a detectable label. Suitable detectable labels and / or moieties that can provide a detectable signal can include, but are not limited to, enzymes, radioisotopes, members of specific binding pairs; fluorophores; fluorescent proteins; quantum dots; and the like.

[0226] Suitable fluorescent proteins include, but are not limited to, green fluorescent protein (GFP) or variants thereof, blue fluorescent variant of GFP (BFP), cyan fluorescent variant of GFP (CFP), yellow fluorescent variant of GFP (YFP), enhanced GFP (EGFP), enhanced CFP (ECFP), enhanced YFP (EYFP), GFPS65T, Emerald, Topaz (TYFP), Venus, Citrine, mCitrine, GFPuv, destabilized EGFP (dEGFP), destabilized ECFP (dECFP), destabilized EYFP (dEYFP), mCFPm, Cerulean, T-Sapphire, CyPet, YPet, mKO, HcRed, t-HcRed, DsRed, DsRed2, DsRed- monomer, J-Red, dimer2, t-dimer2 (12), mRFP1, pocilloporin, Renilla GFP, MonsterGFP, paGFP, Kaede protein and kindling protein, phycobiliproteins and phycobiliprotein conjugates (including B-phycoerythrin, R-phycoerythrin, and allophycocyanin). Other examples of fluorescent proteins include mHoneydew, mBanana, mOrange, dTomato, tdTomato, mTangerine, mStrawberry, mCherry, mGrape1, mRaspberry, mGrape2, mPlum (Shaner et al. (2005) Nat. Methods 2:905-909), and the like. Any of the various fluorescent proteins and colored proteins from the coral species described in, e.g., Matz et al. (1999) Nature Biotechnol. 17:969-973 are suitable for use.

[0227] Suitable enzymes include, but are not limited to, horseradish peroxidase (HRP), alkaline phosphatase (AP), beta-galactosidase (GAL), glucose-6-phosphate dehydrogenase, beta-N-acetylglucosaminidase, beta-glucuronidase, invertase, xanthine oxidase, firefly luciferase, glucose oxidase (GO), and the like.

[0228]

protospacer adjacent motif (PAM)

[0229] A Cas12J protein binds to a target DNA at a target sequence defined by a region of complementarity between the target DNA-targeting RNA and the target DNA. As is the case with many CRISPR endonucleases, site-specific binding (and / or cleavage) of a double-stranded target DNA occurs at a position determined by both: (i) base-pairing complementarity between the guide RNA and the target DNA; and (ii) a short motif in the target DNA [termed a protospacer adjacent motif (PAM)].

[0230] In some embodiments, the PAM for a Cas12J protein is located directly 5' of the target sequence of the non-complementary strand of the target DNA (complementary strand: (i) hybridizes to the guide sequence of the guide RNA, while the non-complementary strand does not directly hybridize to the guide RNA; and (ii) is the reverse complement of the non-complementary strand).

[0231] In some cases (e.g., when using Cas12J-1947455 - also referred to herein as “Ortholog #1” - as described herein), the PAM sequence of the non-complementary strand is 5'- VTTR -3' (where V is G, A, or C and R is A or G) - see, e.g., FIG. 13 Thus, in some cases, suitable PAMs can include GTTA, GTTG, ATTA, ATTG, CTTA, and CTTG.

[0232] In some cases (e.g., when using Cas12J-2071242 - also referred to herein as “Ortholog #2” - as described herein), the PAM sequence of the non-complementary strand is 5'- TBN -3' (where B is T, C, or G) - see, e.g., FIG. 13 Thus, in some cases, suitable PAMs can include TTA, TTC, TTT, TTG, TCA, TCC, TCT, TCG, TGA, TGC, TGT, and TGG. In some embodiments (e.g., when using Cas12J-2071242 - also referred to herein as “Ortholog #2” - as described herein), the PAM sequence of the non-complementary strand is 5'- TNN -3'.

[0233] In some cases (e.g., when using Cas12J-3339380 - also referred to herein as “Ortholog #3” - as described herein), the PAM sequence of the non-complementary strand is 5'- VTTB -3' (where V is G, A, or C and B is T, C, or G) - see, e.g., FIG. 7Therefore, in some cases, suitable PAMs may include GTTT, GTTC, GTTG, ATTT, ATTC, ATTG, CTTT, CTTC, and CTTG. In some cases (e.g., when using Cas12J-3339380 as described herein – also referred to herein as “ortholog #3”), the PAM sequence of the non-complementary strand is 5'-NTTN-3'. In some cases (e.g., when using Cas12J-3339380 as described herein – also referred to herein as “ortholog #3”), the PAM sequence of the non-complementary strand is 5'-VTTN-3' (where V is G, A, or C). In some embodiments (e.g., when using Cas12J-3339380 as described herein – also referred to herein as “ortholog #3”), the PAM sequence of the non-complementary strand is 5'-VTTC-3'.

[0234] In some cases, different Cas12J proteins (i.e., Cas12J proteins from various species) can be advantageously used in various provided methods to utilize the diverse enzymatic characteristics of different Cas12J proteins (e.g., for different PAM sequence preferences; for increased or decreased enzyme activity; for increased or decreased cytotoxicity levels; for balancing changes between NHEJ, homology-directed repair, single-strand breaks, double-strand breaks, etc.; for utilizing short total sequences; etc.). Cas12J proteins from different species may require different PAM sequences in the target DNA. Therefore, for a particular Cas12J protein of choice, the PAM sequence preference may differ from the sequences described above. Various methods for identifying appropriate PAM sequences (including computer simulation methods and / or wet lab methods) are known and conventional in the art, and any convenient method may be used. For example, the PAM sequences described herein were identified using PAM depletion analysis (e.g., see the working examples below), but may also have been identified using a variety of different methods (including computational analysis of sequencing data known in the art).

[0235]

Cas12J guide RNA

[0236] Nucleic acids that bind to the Cas12J protein to form a ribonucleoprotein complex (RNP) and target the complex to a specific location within a target nucleic acid (e.g., target DNA) are referred to herein as “Cas12J guide RNA” or simply “guide RNA”. It should be understood that in some cases, hybrid DNA / RNA can be prepared such that the Cas12J guide RNA includes DNA bases in addition to RNA bases, but the term “Cas12J guide RNA” is still used to encompass such molecules as described herein.

[0237] It can be said that a Casl2J guide RNA comprises two segments, a targeting segment and a protein-binding segment. The protein-binding segment is also referred to herein as the "constant region" of the guide RNA. The targeting segment of a Casl2J guide RNA comprises a nucleotide sequence (guide sequence) that is complementary to (and thus hybridizes with) a particular sequence (target site) within a target nucleic acid (e.g., a target dsDNA, a target ssRNA, a target ssDNA, a complementary strand of a double-stranded target DNA, etc.). The protein-binding segment (or "protein-binding sequence") interacts with (binds to) a Casl2J polypeptide. The protein-binding segment of a subject Casl2J guide RNA can comprise two stretches of complementary nucleotides that hybridize to each other to form a double-stranded RNA duplex (dsRNA duplex). Site-specific binding and / or cleavage of a target nucleic acid (e.g., genomic DNA, dsDNA, RNA, etc.) can occur at a location (e.g., a target sequence of a target locus) determined by base-pairing complementarity between a Casl2J guide RNA (guide sequence of the Casl2J guide RNA) and the target nucleic acid.

[0238] A Casl2J guide RNA and a Casl2J protein (e.g., a wild-type Casl2J protein; a variant Casl2J protein; a fusion Casl2J polypeptide; etc.) form a complex (e.g., bind via non-covalent interactions). The Casl2J guide RNA provides target specificity to the complex by including a targeting segment that includes a guide sequence (a nucleotide sequence that is complementary to a sequence of a target nucleic acid). The Casl2J protein of the complex provides site-specific activity (e.g., cleavage activity provided by the Casl2J protein and / or activity provided by a fusion partner in the case of a fusion Casl2J protein). In other words, the Casl2J protein is directed to a target nucleic acid sequence (e.g., a target sequence) as a result of its association with the Casl2J guide RNA.

[0239] A "guide sequence," also referred to as a "targeting sequence" of a Casl2J guide RNA, can be modified such that the Casl2J guide RNA can target a Casl2J protein (e.g., a naturally-occurring Casl2J protein, a fusion Casl2J polypeptide, etc.) to any desired sequence of any desired target nucleic acid, subject to (e.g., as described herein) consideration of the PAM sequence. Thus, for example, a Casl2J guide RNA can have a guide sequence that is complementary to (e.g., can hybridize with) a sequence in a nucleic acid in a eukaryotic cell, such as a viral nucleic acid, a eukaryotic nucleic acid (e.g., a eukaryotic chromosome, a chromosomal sequence, a eukaryotic RNA, etc.), etc.

[0240]

Guide sequence of a Casl2J guide RNA

[0241] A subject Casl2J guide RNA includes a guide sequence (i.e., a targeting sequence) that is a nucleotide sequence that is complementary to a sequence (a target site) in a target nucleic acid. In other words, the guide sequence of a Casl2J guide RNA can interact with a target nucleic acid (e.g., double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), single-stranded RNA (ssRNA), or double-stranded RNA (dsRNA)) in a sequence-specific manner via hybridization (i.e., base pairing). The guide sequence of a Casl2J guide RNA can be modified (e.g., by genetic engineering) / designed to hybridize to any desired target sequence within a target nucleic acid (e.g., a eukaryotic target nucleic acid, such as genomic DNA) (e.g., when considering a PAM, e.g., when targeting a dsDNA target).

[0242] In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid is 60% or more (e.g., 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%). In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid is 80% or more (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%). In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid is 90% or more (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100%). In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid is 100%.

[0243] In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid is 100% over the seven consecutive nucleotides 3’ most of the target site of the target nucleic acid.

[0244] In some cases, the percentage of complementarity between the guide sequence and the target site of the target nucleic acid is 60% or higher (e.g., 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%) over 17 or more (e.g., 18 or more, 19 or more, 20 or more, 21 or more, 22 or higher) consecutive nucleotides. In other cases, the percentage of complementarity between the guide sequence and the target site of the target nucleic acid is 80% or higher (e.g., 85% or higher, 90% or higher, 95% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%) over 17 or more (e.g., 18 or more, 19 or more, 20 or more, 21 or more, 22 or higher) consecutive nucleotides. In some cases, the percentage of complementarity between the guide sequence and the target site of the target nucleic acid is 90% or higher (e.g., 95%, 97%, 98%, 99%, or 100%) over 17 or more (e.g., 18 or more, 19 or more, 20 or more, 21 or more, 22 or more) consecutive nucleotides. In other cases, the percentage of complementarity between the guide sequence and the target site of the target nucleic acid is 100% over 17 or more (e.g., 18 or more, 19 or more, 20 or more, 21 or more, 22 or more) consecutive nucleotides.

[0245] In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid is 60% or greater (e.g., 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 97% or greater, 98% or greater, 99% or greater, or 100%) over 19 or more (e.g., 20 or more, 21 or more, 22 or more) contiguous nucleotides. In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid is 80% or greater (e.g., 85% or greater, 90% or greater, 95% or greater, 97% or greater, 98% or greater, 99% or greater, or 100%) over 19 or more (e.g., 20 or more, 21 or more, 22 or more) contiguous nucleotides. In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid is 90% or greater (e.g., 95% or greater, 97% or greater, 98% or greater, 99% or greater, or 100%) over 19 or more (e.g., 20 or more, 21 or more, 22 or more) contiguous nucleotides. In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid is 100% over 19 or more (e.g., 20 or more, 21 or more, 22 or more) contiguous nucleotides.

[0246] In some embodiments, the percent complementarity between the guide sequence and the target site of the target nucleic acid is 60% or greater (e.g., 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 97% or greater, 98% or greater, 99% or greater, or 100%) over 17-25 contiguous nucleotides. In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid is 80% or greater (e.g., 85% or greater, 90% or greater, 95% or greater, 97% or greater, 98% or greater, 99% or greater, or 100%) over 17-25 contiguous nucleotides. In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid is 90% or greater (e.g., 95% or greater, 97% or greater, 98% or greater, 99% or greater, or 100%) over 17-25 contiguous nucleotides. In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid is 100% over 17-25 contiguous nucleotides.

[0247] In some embodiments, the percent complementarity between the guide sequence and the target site of the target nucleic acid is 60% or more (e.g., 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) over 19-25 contiguous nucleotides. In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid is 80% or more (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) over 19-25 contiguous nucleotides. In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid is 90% or more (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100%) over 19-25 contiguous nucleotides. In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid is 100% over 19-25 contiguous nucleotides.

[0248] In some cases, the guide sequence has a length in the range of 17-30 nucleotides (nt) (e.g., 17-25, 17-22, 17-20, 19-30, 19-25, 19-22, 19-20, 20-30, 20-25, or 20-22 nt). In some cases, the guide sequence has a length in the range of 17-25 nucleotides (nt) (e.g., 17-22, 17-20, 19-25, 19-22, 19-20, 20-25, or 20-22 nt). In some cases, the guide sequence has a length of 17 or more nt (e.g., 18 or more, 19 or more, 20 or more, 21 or more, or 22 or more nt; 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, etc.). In some cases, the guide sequence has a length of 19 or more nt (e.g., 20 or more, 21 or more, or 22 or more nt; 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, etc.). In some cases, the guide sequence has a length of 17 nt. In some cases, the guide sequence has a length of 18 nt. In some cases, the guide sequence has a length of 19 nt. In some cases, the guide sequence has a length of 20 nt. In some cases, the guide sequence has a length of 21 nt. In some cases, the guide sequence has a length of 22 nt. In some cases, the guide sequence has a length of 23 nt.

[0249] In some cases, the guide sequence (also referred to as a “spacer sequence”) has a length of 15 to 50 nucleotides (e.g., 15 nucleotides (nt) to 20 nt, 20 nt to 25 nt, 25 nt to 30 nt, 30 nt to 35 nt, 35 nt to 40 nt, 40 nt to 45 nt, or 45 nt to 50 nt).

[0250] Protein-binding segment of a Cas12J guide RNA

[0251] The protein-binding segment of a subject Cas12J guide RNA (“constant region”) interacts with a Cas12J protein. The Cas12J guide RNA directs the bound Cas12J protein to a specific nucleotide sequence within a target nucleic acid via the guide sequence mentioned above. The protein-binding segment of a Cas12J guide RNA can include two stretches of nucleotides that are complementary to each other and hybridize to form a double-stranded RNA duplex (dsRNA duplex). Thus, in some cases, the protein-binding segment includes a dsRNA duplex.

[0252] In some cases, the dsRNA duplex region comprises a range of 5-25 base pairs (bp) (e.g., 5-22, 5-20, 5-18, 5-15, 5-12, 5-10, 5-8, 8-25, 8-22, 8-18, 8-15, 8-12, 12-25, 12-22, 12-18, 12-15, 13-25, 13-22, 13-18, 13-15, 14-25, 14-22, 14-18, 14-15, 15-25, 15-22, 15-18, 17-25, 17-22, or 17-18 bp, e.g., 5 bp, 6 bp, 7 bp, 8 bp, 9 bp, 10 bp, etc.). In some cases, the dsRNA duplex region comprises a range of 6-15 base pairs (bp) (e.g., 6-12, 6-10, or 6-8 bp, e.g., 6 bp, 7 bp, 8 bp, 9 bp, 10 bp, etc.). In some cases, the duplex region comprises 5 or more bp (e.g., 6 or more, 7 or more, or 8 or more bp). In some cases, the duplex region comprises 6 or more bp (e.g., 7 or more, or 8 or more bp). In some cases, not all nucleotides of the duplex region are paired, and thus the duplex forming region can include bulges. The term “bulge” herein is used to mean a stretch of nucleotides (which can be one nucleotide) that does not contribute to the double-stranded duplex, but is surrounded by contributing nucleotides at the 5’ and 3’, and thus the bulge is considered part of the duplex region. In some cases, the dsRNA comprises 1 or more bulges (e.g., 2 or more, 3 or more, 4 or more bulges). In some cases, the dsRNA duplex comprises 2 or more bulges (e.g., 3 or more, 4 or more bulges). In some cases, the dsRNA duplex comprises 1-5 bulges (e.g., 1-4, 1-3, 2-5, 2-4, or 2-3 bulges).

[0253] Accordingly, in some cases, the stretches of nucleotides that hybridize to each other to form a dsRNA duplex have 70-100% complementarity to each other (e.g., 75-100%, 80-10%, 85-100%, 90-100%, 95-100% complementarity). In some cases, the stretches of nucleotides that hybridize to each other to form a dsRNA duplex have 70-100% complementarity to each other (e.g., 75-100%, 80-10%, 85-100%, 90-100%, 95-100% complementarity). In some cases, the stretches of nucleotides that hybridize to each other to form a dsRNA duplex have 85-100% complementarity to each other (e.g., 90-100%, 95-100% complementarity). In some cases, the stretches of nucleotides that hybridize to each other to form a dsRNA duplex have 70-95% complementarity to each other (e.g., 75-95%, 80-95%, 85-95%, 90-95% complementarity).

[0254] Accordingly, in some cases, the stretches of nucleotides that hybridize to each other to form a dsRNA duplex have 70-100% complementarity to each other (e.g., 75-100%, 80-10%, 85-100%, 90-100%, 95-100% complementarity). In some cases, the stretches of nucleotides that hybridize to each other to form a dsRNA duplex have 70-100% complementarity to each other (e.g., 75-100%, 80-10%, 85-100%, 90-100%, 95-100% complementarity). In some cases, the stretches of nucleotides that hybridize to each other to form a dsRNA duplex have 85-100% complementarity to each other (e.g., 90-100%, 95-100% complementarity). In some cases, the stretches of nucleotides that hybridize to each other to form a dsRNA duplex have 70-95% complementarity to each other (e.g., 75-95%, 80-95%, 85-95%, 90-95% complementarity).

[0255] The duplex region of a subject Casl2J guide RNA can comprise one or more (1, 2, 3, 4, 5, etc.) mutations relative to a naturally occurring duplex region. For example, in some cases, the base pairs can be maintained while the nucleotides contributing to the base pairs of each segment can be different. In some cases, the duplex region of a subject Casl2J guide RNA comprises more paired bases, fewer paired bases, a smaller bulge, a larger bulge, fewer bulges, more bulges, or any convenient combination thereof, as compared to a naturally occurring duplex region (of a naturally occurring Casl2J guide RNA).

[0256] Examples of various Cas9 guide RNAs can be found in the art, and in some cases, similar variations to those introduced into Cas9 guide RNAs can also be introduced into the Cas12J guide RNAs of the present disclosure (e.g., mutations of the dsRNA duplex region, extensions of the 5' or 3' ends for increased stability to provide interactions with another protein, etc.). See, e.g., Jinek et al., Science. 2012 Aug 17;337(6096):816-21; Chylinski et al., RNA Biol. 2013 May;10(5):726-37; Ma et al., Biomed Res Int. 2013;2013:270805; Hou et al., Proc Natl Acad Sci U S A. 2013 Sep 24;110(39):15644-9; Jinek et al., Elife. 2013;2:e00471; Pattanayak et al., Nat Biotechnol. 2013 Sep;31(9):839-43; Qi et al., Cell. 2013 Feb 28;152(5):1173-83; Wang et al., Cell. 2013 May 9;153(4):910-8; Auer et al., Genome Res. 2013 Oct 31; Chen et al., Nucleic Acids Res. 2013 Nov 1;41(20):e19; Cheng et al., Cell Res. 2013 Oct;23(10):1163-71; Cho et al., Genetics. 2013 Nov;195(3):1177-80; DiCarlo et al., Nucleic Acids Res. 2013 Apr;41(7):4336-43; Dickinson et al., Nat Methods. 2013 Oct;10(10):1028-34; Ebina et al., Sci Rep. 2013;3:2510; Fujii et al., Nucleic Acids Res. 2013 Nov 1;41(20):e187; Hu et al., Cell Res. 2013 Nov;23(11):1322-5; Jiang et al., Nucleic Acids Res. 2013 Nov 1;41(20):e188; Larson et al., Nat Protoc. 2013 Nov;8(11):2180-96; Mali et al., Nat Methods. 2013 Oct;10(10):957-63; Nakayama et al., Genesis.2013 Dec;51(12):835-43; Ran et al., Nat Protoc. 2013 Nov;8(11):2281-308; Ran et al., Cell. 2013 Sep 12;154(6): 1380-9; Upadhyay et al., G3 (Bethesda). 2013 Dec 9;3(12):2233-8; Walsh et al., Proc Natl Acad Sci U S A. 2013 Sep 24;110(39):15514-5; Xie et al., Mol Plant. 2013 Oct 9; Yang et al., Cell. 2013 Sep 12;154(6): 1370-9; Briner et al., Mol Cell.October 23, 2014; 56(2): 333-9; and the following U.S. patents and patent applications: 8,906,616; 8,895,308; 8,889,418; 8,889,356; 8,871,445; 8,865,406; 8,795,965; 8,771,945; 8,697,359; 20140068797; 20140170753; 20140179006; 20140179770; 20140186843; 20140186919; 20140186958; 20140189896; 20140227787; 20140234972; 20140242664; 20140242699; 20140242700; 20140242702; 20140248702; 20140256046; 20140273037; 20140273226; 20140273230; 20140273231; 20140273232; 20140273233; 20140273234; 20140273235; 20140287938; 20140295556; 20140295557; 20140298547; 20140304853; 20140309487; 20140310828; 20140310830; 20140315985; 20140335063; 20140335620; 20140342456; 20140342457; 20140342458; 20140349400; 20140349405; 20140356867; 20140356956; 20140356958; 20140356959; 20140357523; 20140357530; 20140364333; and 20140377868; which are all hereby incorporated by reference in their entirety.

[0257] Examples of constant regions suitable for inclusion in a Cas12J guide RNA are provided in FIG. 7 ( e.g., where T is substituted with U). The Cas12J guide RNA depicted in FIG. 8GUCUCGACUAAUCGAGCAAU CGUUUGAGAU CUCUCC (SEQ ID NO: 83). As another example, the constant region of a Casl2J guide RNA can comprise the nucleotide sequence: GUCGGAACGC UCAACGAUUG CCCCUCACGA GGGGAC (SEQ ID NO: 84). As another example, the constant region of a Casl2J guide RNA can comprise the nucleotide sequence: GUCCCAGCGU ACUGGGCAAUCAAUAGTCGU UUUGGU (SEQ ID NO: 85). As another example, the constant region of a Casl2J guide RNA can comprise the nucleotide sequence: CACAGGAGAG AUCUCAAACG AUUGCUCGAU UAGUCGAGAC (SEQ ID NO: 86). As another example, the constant region of a Casl2J guide RNA can comprise the nucleotide sequence: UAAUGUCGGA ACGCUCAACG AUUGCCCCUCACGAGGGGAC (SEQ ID NO: 87). As another example, the constant region of a Casl2J guide RNA can comprise the nucleotide sequence: AUUAACCAAA ACGACUAUUG AUUGCCCAGU ACGCUGGGAC (SEQ ID NO: 88).

[0258] The Casl2J guide RNA constant region can comprise any of the nucleotide sequences depicted in FIG. 8 The Casl2J guide RNA constant region can comprise a nucleotide sequence within the consensus sequence depicted in FIG. 7

[0259] The nucleotide sequence (in which T is substituted with U) can be combined with a selected spacer sequence (in which the spacer sequence comprises a target nucleic acid binding sequence (“guide sequence”)) that is 15 to 50 nucleotides (e.g., 15 nucleotides (nt) to 20 nt, 20 nt to 25 nt, 25 nt to 30 nt, 30 nt to 35 nt, 35 nt to 40 nt, 40 nt to 45 nt, or 45 nt to 50 nt long). In some cases, the spacer sequence is 35-38 nucleotides long. For example, the nucleotide sequence depicted in FIG. 7 ​The reverse complement of any of the nucleotide sequences in (a) (but with T replaced by U) can be included in a guide RNA comprising a constant region-(N)n, where N is any nucleotide and n is an integer from 15 to 50 (e.g., 15 to 20, 20 to 25, 25 to 30, 30 to 35, 35 to 38, 35 to 40, 40 to 45, or 45 to 50). FIG. 6A-6R The reverse complement of any of the nucleotide sequences in (a) (but with T replaced by U) can be included in a guide RNA comprising a constant region-(N)n, where N is any nucleotide and n is an integer from 15 to 50 (e.g., 15 to 20, 20 to 25, 25 to 30, 30 to 35, 35 to 38, 35 to 40, 40 to 45, or 45 to 50).

[0260] As one example, a guide RNA can have the following nucleotide sequence: NNNNNNNNNNN NNNNNNNNNNN NNNNNNNNNNN NNNNNGUCUC GACUAAUCGA GCAAUCGUUU GAGAUCUCUC C (SEQ ID NO: 89) or in some cases the reverse complement, where N is any nucleotide, e.g., where the Ns segment comprises a target nucleic acid binding sequence. As another example, a guide RNA can have the following nucleotide sequence: NNNNNNNNNNN NNNNNNNNNN NNNNNNNNNNN NNNNNGUCGG AACGCUCAAC GAUUGCCCCU CACGAGGGGA C (SEQ ID NO: 90) or in some cases the reverse complement, where N is any nucleotide, e.g., where the Ns segment comprises a target nucleic acid binding sequence.

[0261] As one example, a guide RNA can have the following nucleotide sequence: GUCUCGACUA AUCGAGCAAUCGUUUGAGAU CUCUCC-‘guide sequence’ (e.g., GUCUCGACUA AUCGAGCAAU CGUUUGAGAU CUCUCCNNNNNNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN N (SEQ ID NO: 91), where the Ns segment represents the guide sequence / targeting sequence and N is any nucleotide). As another example, a guide RNA can have the following nucleotide sequence: GGAGAGAUCUCAAACGAUUG CUCGAUUAGU CGAGAC-‘guide sequence’ (e.g., GGAGAGAUCU CAAACGAUUG CUCGAUUAGU CGAGACNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN N (SEQ ID NO: 92), where the Ns segment represents the guide sequence / targeting sequence and N is any nucleotide).

[0262] As another example, a guide RNA can have the following nucleotide sequence: GUCGGAACGC UCAACGAUUG CCCCUCACGA GGGGAC - guide sequence (e.g., GUCGGAACGC UCAACGAUUG CCCCUCACGA GGGGACNNNNNNNNNNNNNN NNNNNNNNNN NNNNNNNNNN N (SEQ ID NO: 93), where the Ns segment represents the guide sequence / targeting sequence and N is any nucleotide). As another example, a guide RNA can have the following nucleotide sequence: GUCCCCUCGUGAGGGGCAAU CGUUGAGCGU UCCGAC - guide sequence (e.g., GUCCCCUCGU GAGGGGCAAU CGUUGAGCGU UCCGACNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN N (SEQ ID NO: 94), where the Ns segment represents the guide sequence / targeting sequence and N is any nucleotide).

[0263] As another example, a guide RNA can have the following nucleotide sequence: CACAGGAGAG AUCUCAAACG AUUGCUCGAU UAGUCGAGAC - guide sequence (e.g., CACAGGAGAG AUCUCAAACG AUUGCUCGAU UAGUCGAGAC NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNN (SEQ ID NO: 95), where the Ns segment represents the guide sequence / targeting sequence and N is any nucleotide). As another example, a guide RNA can have the following nucleotide sequence: UAAUGUCGGA ACGCUCAACG AUUGCCCCUC ACGAGGGGAC - guide sequence (e.g., UAAUGUCGGAACGCUCAACG AUUGCCCCUC ACGAGGGGAC NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNN (SEQ ID NO: 96), where the Ns segment represents the guide sequence / targeting sequence and N is any nucleotide). As another example, a guide RNA can have the following nucleotide sequence: AUUAACCAAA ACGACUAUUG AUUGCCCAGU ACGCUGGGAC - guide sequence (e.g., AUUAACCAAAA CGACUAUUG AUUGCCCAGU ACGCUGGGAC NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNN (SEQ ID NO: 97), where the Ns segment represents the guide sequence / targeting sequence and N is any nucleotide).

[0264]

Cas12J guide polynucleotide

[0265] In some cases, a nucleic acid binds to a Casl2J protein, thereby forming a nucleic acid / Casl2J polypeptide complex, and targets the complex to a particular location within a target nucleic acid (e.g., a target DNA). In some cases, the guide polynucleotide comprises ribonucleotides alone, deoxyribonucleotides alone, or a mixture of ribonucleotides and deoxyribonucleotides. In some cases, the guide polynucleotide comprises ribonucleotides alone, and is referred to herein as a "guide RNA." In some cases, the guide polynucleotide comprises deoxyribonucleotides alone, and is referred to herein as a "guide DNA." In some cases, the guide polynucleotide comprises both ribonucleotides and deoxyribonucleotides. A guide polynucleotide can comprise combinations of ribonucleotide bases, deoxyribonucleotide bases, nucleotide analogs, modified nucleotides, etc.; and can also include naturally-occurring backbone residues and / or linkages and / or non-naturally-occurring backbone residues and / or linkages.

[0266]

Cas12J system

[0267] This disclosure provides a Cas12J system. The Cas12J system of this disclosure may comprise: a) the Cas12J peptide and Cas12J guide RNA of this disclosure; b) the Cas12J peptide, Cas12J guide RNA, and donor template nucleic acid of this disclosure; c) the Cas12J fusion peptide and Cas12J guide RNA of this disclosure; d) the Cas12J fusion peptide, Cas12J guide RNA, and donor template nucleic acid of this disclosure; e) mRNA encoding the Cas12J peptide of this disclosure; and Cas12J guide RNA; f) mRNA encoding the Cas12J peptide of this disclosure, Cas12J guide RNA, and donor template nucleic acid; g) encoding the C... h) mRNA encoding the Cas12J fusion peptide disclosed herein, Cas12J guide RNA, and donor template nucleic acid; i) a recombinant expression vector comprising a nucleotide sequence encoding the Cas12J peptide disclosed herein and a nucleotide sequence encoding the Cas12J guide RNA; j) a recombinant expression vector comprising a nucleotide sequence encoding the Cas12J peptide disclosed herein, a nucleotide sequence encoding the Cas12J guide RNA, and a nucleotide sequence encoding the donor template nucleic acid; k) a recombinant expression vector comprising a nucleotide sequence encoding the Cas12J fusion peptide disclosed herein and a nucleotide sequence encoding the donor template nucleic acid; The expression vector comprises: a nucleotide sequence encoding the Cas12J guide RNA; a recombinant expression vector comprising a nucleotide sequence encoding the Cas12J fusion polypeptide disclosed herein, a nucleotide sequence encoding the Cas12J guide RNA, and a nucleotide sequence encoding a donor template nucleic acid; a first recombinant expression vector comprising a nucleotide sequence encoding the Cas12J polypeptide disclosed herein, and a second recombinant expression vector comprising a nucleotide sequence encoding the Cas12J guide RNA; a first recombinant expression vector comprising a nucleotide sequence encoding the Cas12J polypeptide disclosed herein, and a second recombinant expression vector comprising a nucleotide sequence encoding the Cas12J guide RNA; and a donor template. Nucleic acid; o) a first recombinant expression vector comprising a nucleotide sequence encoding the Cas12J fusion polypeptide of the present invention, and a second recombinant expression vector comprising a nucleotide sequence encoding a Cas12J guide RNA; p) a first recombinant expression vector comprising a nucleotide sequence encoding the Cas12J fusion polypeptide of the present invention, and a second recombinant expression vector comprising a nucleotide sequence encoding a Cas12J guide RNA; and donor template nucleic acid; q) a recombinant expression vector comprising a nucleotide sequence encoding the Cas12J polypeptide of the present invention, a nucleotide sequence encoding a first Cas12J guide RNA, and a nucleotide sequence encoding a second Cas12J guide RNA;or r) a recombinant expression vector comprising a nucleotide sequence encoding a Cas12J fusion polypeptide of the disclosure, a nucleotide sequence encoding a first Cas12J guide RNA, and a nucleotide sequence encoding a second Cas12J guide RNA; or a variant of any of (a) to (r).

[0268] [NUCLEIC ACIDS]

[0269] The disclosure provides one or more nucleic acids comprising one or more of: a donor polynucleotide sequence, a nucleotide sequence encoding a Cas12J polypeptide (e.g., a wild-type Cas12J protein, a nickase Cas12J protein, a dCas12J protein, a fusion Cas12J protein, etc.), a Cas12J guide RNA, and a nucleotide sequence encoding a Cas12J guide RNA. The disclosure provides a nucleic acid comprising a nucleotide sequence encoding a Cas12J fusion polypeptide. The disclosure provides a recombinant expression vector comprising a nucleotide sequence encoding a Cas12J polypeptide. The disclosure provides a recombinant expression vector comprising a nucleotide sequence encoding a Cas12J fusion polypeptide. The disclosure provides a recombinant expression vector comprising: a) a nucleotide sequence encoding a Cas12J polypeptide; and b) a nucleotide sequence encoding a Cas12J guide RNA. The disclosure provides a recombinant expression vector comprising: a) a nucleotide sequence encoding a Cas12J fusion polypeptide; and b) a nucleotide sequence encoding a Cas12J guide RNA. In some cases, the nucleotide sequence encoding a Cas12J protein and / or the nucleotide sequence encoding a Cas12J guide RNA is operably linked to a promoter that is operable in a selected cell type (e.g., a prokaryotic cell, a eukaryotic cell, a plant cell, an animal cell, a mammalian cell, a primate cell, a rodent cell, a human cell, etc.).

[0270] In some cases, the nucleotide sequence encoding a Cas12J polypeptide of the disclosure is codon-optimized. This type of optimization can entail mutation of the nucleotide sequence encoding Cas12J to mimic the codon bias of the intended host organism or cell while encoding the same protein. Thus, the codons can vary, but the encoded protein remains the same. For example, if the intended target cell is a human cell, then a human codon-optimized nucleotide sequence encoding Cas12J can be used. As another non-limiting example, if the intended host cell is a mouse cell, then a mouse codon-optimized nucleotide sequence encoding Cas12J can be generated. As another non-limiting example, if the intended host cell is a plant cell, then a plant codon-optimized nucleotide sequence encoding Cas12J can be generated. As another non-limiting example, if the intended host cell is an insect cell, then an insect codon-optimized nucleotide sequence encoding Cas12J can be generated.

[0271] A table of codon usage is readily available, for example, in the "Codon Usage Database" available at www.kazusa.or.jp / codon. In some cases, a nucleic acid of the present disclosure comprises a nucleotide sequence encoding a Casl2J polypeptide that is codon-optimized for expression in a eukaryotic cell. In some cases, a nucleic acid of the present disclosure comprises a nucleotide sequence encoding a Casl2J polypeptide that is codon-optimized for expression in an animal cell. In some cases, a nucleic acid of the present disclosure comprises a nucleotide sequence encoding a Casl2J polypeptide that is codon-optimized for expression in a fungal cell. In some cases, a nucleic acid of the present disclosure comprises a nucleotide sequence encoding a Casl2J polypeptide that is codon-optimized for expression in a plant cell. In some cases, a nucleic acid of the present disclosure comprises a nucleotide sequence encoding a Casl2J polypeptide that is codon-optimized for expression in a monocot plant species. In some cases, a nucleic acid of the present disclosure comprises a nucleotide sequence encoding a Casl2J polypeptide that is codon-optimized for expression in a dicot plant species. In some cases, a nucleic acid of the present disclosure comprises a nucleotide sequence encoding a Casl2J polypeptide that is codon-optimized for expression in a gymnosperm plant species. In some cases, a nucleic acid of the present disclosure comprises a nucleotide sequence encoding a Casl2J polypeptide that is codon-optimized for expression in an angiosperm plant species. In some cases, a nucleic acid of the present disclosure comprises a nucleotide sequence encoding a Casl2J polypeptide that is codon-optimized for expression in a corn cell. In some cases, a nucleic acid of the present disclosure comprises a nucleotide sequence encoding a Casl2J polypeptide that is codon-optimized for expression in a soybean cell. In some cases, a nucleic acid of the present disclosure comprises a nucleotide sequence encoding a Casl2J polypeptide that is codon-optimized for expression in a rice cell. In some cases, a nucleic acid of the present disclosure comprises a nucleotide sequence encoding a Casl2J polypeptide that is codon-optimized for expression in a wheat cell. In some cases, a nucleic acid of the present disclosure comprises a nucleotide sequence encoding a Casl2J polypeptide that is codon-optimized for expression in a cotton cell. In some cases, a nucleic acid of the present disclosure comprises a nucleotide sequence encoding a Casl2J polypeptide that is codon-optimized for expression in a sorghum cell. In some cases, a nucleic acid of the present disclosure comprises a nucleotide sequence encoding a Casl2J polypeptide that is codon-optimized for expression in an alfalfa cell. In some cases, a nucleic acid of the present disclosure comprises a nucleotide sequence encoding a Casl2J polypeptide that is codon-optimized for expression in a sugarcane cell. In some cases, a nucleic acid of the present disclosure comprises a nucleotide sequence encoding a Casl2J polypeptide that is codon-optimized for expression in an Arabidopsis cell.In some cases, a nucleic acid of the present disclosure comprises a nucleotide sequence encoding a Casl2J polypeptide that is codon-optimized for expression in a tomato cell. In some cases, a nucleic acid of the present disclosure comprises a nucleotide sequence encoding a Casl2J polypeptide that is codon-optimized for expression in a cucumber cell. In some cases, a nucleic acid of the present disclosure comprises a nucleotide sequence encoding a Casl2J polypeptide that is codon-optimized for expression in a potato cell. In some cases, a nucleic acid of the present disclosure comprises a nucleotide sequence encoding a Casl2J polypeptide that is codon-optimized for expression in an algal cell.

[0272] The present disclosure provides one or more recombinant expression vectors comprising (in some cases in different recombinant expression vectors, and in some cases in the same recombinant expression vector): (i) a nucleotide sequence of a donor template nucleic acid (wherein the donor template comprises a nucleotide sequence having homology to a target sequence of a target nucleic acid (e.g., a target genome); (ii) a nucleotide sequence encoding a Casl2J guide RNA that hybridizes to a target sequence of a target locus of the targeted genome (e.g., operably linked to a promoter operable in a target cell such as a eukaryotic cell); and (iii) a nucleotide sequence encoding a Casl2J protein (e.g., operably linked to a promoter operable in a target cell such as a eukaryotic cell). The present disclosure provides one or more recombinant expression vectors comprising (in some cases in different recombinant expression vectors, and in some cases in the same recombinant expression vector): (i) a nucleotide sequence of a donor template nucleic acid (wherein the donor template comprises a nucleotide sequence having homology to a target sequence of a target nucleic acid (e.g., a target genome); and (ii) a nucleotide sequence encoding a Casl2J guide RNA that hybridizes to a target sequence of a target locus of the targeted genome (e.g., operably linked to a promoter operable in a target cell such as a eukaryotic cell). The present disclosure provides one or more recombinant expression vectors comprising (in some cases in different recombinant expression vectors, and in some cases in the same recombinant expression vector): (i) a nucleotide sequence encoding a Casl2J guide RNA that hybridizes to a target sequence of a target locus of the targeted genome (e.g., operably linked to a promoter operable in a target cell such as a eukaryotic cell); and (ii) a nucleotide sequence encoding a Casl2J protein (e.g., operably linked to a promoter operable in a target cell such as a eukaryotic cell).

[0273] Suitable expression vectors include viral expression vectors (e.g., viral vectors based on the following viruses: vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO 94 / 12649, WO 93 / 03769; WO 93 / 19191; WO 94 / 28938; WO 95 / 11984; and WO 95 / 00655); adeno-associated virus (AAV) (see, e.g., Ali et al., Hum Gene Ther 9:81 86, 1998; Flannery et al., PNAS 94:6916 6921, 1997; Bennett et al., Invest Opthalmol Vis Sci 38:2857 2863, 1997; Jomary et al., Gene Ther 4:683 690, 1997; Rolling et al., Hum Gene Ther 10:641 648, 1999; Ali et al., Hum Mol Genet 5:591 594, 1996; WO 93 / 09239 to Srivastava; Samulski et al., J. Vir. (1989) 63:3822-3828; Mendelson et al., Virol. (1988) 166:154-165; and Flotte et al., PNAS (1993) 90:10613-10617); SV40; herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshi et al., PNAS 94:10319 23, 1997; Takahashi et al., J Virol 73:7812 7816, 1999); retroviral vectors (e.g., murine leukemia virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, lentivirus, human immunodeficiency virus, myeloblastosis sarcoma virus, and mammary tumor virus), and the like. In some cases, the recombinant expression vector of the present disclosure is a recombinant adeno-associated virus (AAV) vector. In some cases, the recombinant expression vector of the present disclosure is a recombinant lentivirus vector. In some cases, the recombinant expression vector of the present disclosure is a recombinant retrovirus vector.

[0274] For plant applications, viral vectors based on a Tobamovirus, a Potexvirus, a Potyvirus, a Tobravirus, a Tombusvirus, a Geminivirus, a Bromovirus, a Carmovirus, a Hypovirus, or a Cucumovirus can be used. See, e.g., Peyret and Lomonossoff (2015) Plant Biotechnol. J. 13:1121. Suitable Tobamovirus vectors include, e.g., a Tomato mosaic virus (ToMV) vector, a Tobacco mosaic virus (TMV) vector, a Tobacco mild green mosaic virus (TMGMV) vector, a Pepper mild mosaic virus (PMMoV) vector, a Pepper powdery mild mosaic virus (PaMMV) vector, a Cucumber green mottle mosaic virus (CGMMV) vector, a Kyuri green mottle mosaic virus (KGMMV) vector, a Hibiscus latent Petersburg virus (HLFPV) vector, an Odontoglossum ringspot virus (ORSV) vector, a Rehmannia mosaic virus (ReMV) vector, a Cactus desiccation-associated virus (SOV) vector, a Wasabi mosaic virus (WMoV) vector, a Oilseed rape mosaic virus (YoMV) vector, a Sida herbivora mosaic virus (SHMV) vector, and the like. Suitable Potexvirus vectors include, e.g., a Potato virus X (PVX) vector, a Potato aucuba mosaic virus (PAMV) vector, a Alsine X virus (AlsVX) vector, a Cactus virus X (CVX) vector, a Cymbidium mosaic virus (CymMV) vector, a Hosta virus X (HVX) vector, a Lily virus X (LVX) vector, a Narcissus mosaic virus (NMV) vector, a Nilon virus X (NVX) vector, a Plantago mosaic virus (PlAMV) vector, a Strawberry mild yellow edge virus (SMYEV) vector, a Tulip virus X (TVX) vector, a White clover mosaic virus (WCiMV) vector, a Bamboo mosaic virus (BaMV) vector, and the like. Suitable Potyvirus vectors include, e.g., a Potato virus Y (PVY) vector, a Bean common mosaic virus (BCMV) vector, a Clover yellow vein virus (ClYVV) vector, a East Asian passion fruit virus (EAPV) vector, a Freesia mosaic virus (FreMV) vector, a Japanese yam mosaic virus (JYMV) vector, a Lettuce mosaic virus (LMV) vector, a Maize dwarf mosaic virus (MDMV) vector, an Onion yellow dwarf virus (OYDV) vector, a Papaya ringspot virus (PRSV) vector, a Pepper mottle virus (PepMoV) vector, a Perilla mottle virus (PerMoV) vector, a Plum pox virus (PPV) vector, a Potato virus A (PVA) vector, a Sorghum mosaic virus (SrMV) vector, a Soybean mosaic virus (SMV) vector, a Sugarcane mosaic virus (SCMV) vector, a Tulip mosaic virus (TulMV) vector, a Turnip mosaic virus (TuMV) vector, a Watermelon mosaic virus (WMV) vector, a Zucchini yellow mosaic virus (ZYMV) vector, a Tobacco etch virus (TEV) vector, and the like.Suitable Tobamovirus vectors include, for example, a tobacco mosaic virus (TMV) vector, and the like. Suitable Tombusvirus vectors include, for example, a tomato bushy stunt virus (TBSV) vector, a pepper mild mottle virus (PMMV) vector, a zucchini mosaic virus (ZMV) vector, a zucchini lethal chlorosis virus (ZLCV) vector, a zucchini yellow mosaic virus (ZYMV) vector, a tomato aspermy virus (TAV) vector, a melon chlorotic leaf 17 virus (MCL17V) vector, a melon chlorotic leaf 18 virus (MCL18V) vector, a melon chlorotic leaf 19 virus (MCL19V) vector, a melon chlorotic leaf 20 virus (MCL20V) vector, a melon chlorotic leaf 21 virus (MCL21V) vector, a melon chlorotic leaf 22 virus (MCL22V) vector, a melon chlorotic leaf 23 virus (MCL23V) vector, a melon chlorotic leaf 24 virus (MCL24V) vector, a melon chlorotic leaf 25 virus (MCL25V) vector, a melon chlorotic leaf 26 virus (MCL26V) vector, a melon chlorotic leaf 27 virus (MCL27V) vector, a melon chlorotic leaf 28 virus (MCL28V) vector, a melon chlorotic leaf 29 virus (MCL29V) vector, a melon chlorotic leaf 30 virus (MCL30V) vector, a melon chlorotic leaf 31 virus (MCL31V) vector, a melon chlorotic leaf 32 virus (MCL32V) vector, a melon chlorotic leaf 33 virus (MCL33V) vector, a melon chlorotic leaf 34 virus (MCL34V) vector, a melon chlorotic leaf 35 virus (MCL35V) vector, a melon chlorotic leaf 36 virus (MCL36V) vector, a melon chlorotic leaf 37 virus (MCL37V) vector, a melon chlorotic leaf 38 virus (MCL38V) vector, a melon chlorotic leaf 39 virus (MCL39V) vector, a melon chlorotic leaf 40 virus (MCL40V) vector, a melon chlorotic leaf 41 virus (MCL41V) vector, a melon chlorotic leaf 42 virus (MCL42V) vector, a melon chlorotic leaf 43 virus (MCL43V) vector, a melon chlorotic leaf 44 virus (MCL44V) vector, a melon chlorotic leaf 45 virus (MCL45V) vector, a melon chlorotic leaf 46 virus (MCL46V) vector, a melon chlorotic leaf 47 virus (MCL47V) vector, a melon chlorotic leaf 48 virus (MCL48V) vector, a melon chlorotic leaf 49 virus (MCL49V) vector, a melon chlorotic leaf 50 virus (MCL50V) vector, and the like. Suitable Pepino mosaic virus vectors include, for example, a Pepino mosaic virus (PepMV) vector, and the like.

[0275] Depending on the host / vector system utilized, any of a wide variety of suitable transcription and translation control elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, and the like can be used in the expression vectors.

[0276] In some embodiments, the nucleotide sequence encoding the Cas12J guide RNA is operably linked to a control element, e.g., a transcription control element, such as a promoter. In some embodiments, the nucleotide sequence encoding the Cas12J protein or Cas12J fusion polypeptide is operably linked to a control element, e.g., a transcription control element, such as a promoter.

[0277] The transcription control element can be a promoter. In some cases, the promoter is a constitutively active promoter. In some cases, the promoter is a regulatable promoter. In some cases, the promoter is an inducible promoter. In some cases, the promoter is a tissue-specific promoter. In some cases, the promoter is a cell-type specific promoter. In some cases, the transcription control element (e.g., promoter) is functional in the targeted cell type or targeted cell population. For example, in some cases, the transcription control element can be functional in eukaryotic cells (e.g., hematopoietic stem cells (e.g., mobilized peripheral blood (mPB) CD34(+) cells, bone marrow (BM) CD34(+) cells, and the like)).

[0278] Non-limiting examples of eukaryotic promoters (promoters that are functional in eukaryotic cells) include those from EF1a, cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, early and late SV40, long terminal repeat (LTR) of retroviruses, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art. The expression vector can also contain a ribosome binding site and a transcription terminator for translation initiation. The expression vector can also include appropriate sequences for amplification of expression. The expression vector can also include a nucleotide sequence encoding a protein tag (e.g., 6xHis tag, hemagglutinin tag, fluorescent protein, etc.) that can be fused to the Cas12J protein, resulting in a fusion Cas12J polypeptide.

[0279] In some embodiments, the nucleotide sequence encoding the Cas12J guide RNA and / or Cas12J fusion polypeptide is operably linked to an inducible promoter. In some embodiments, the nucleotide sequence encoding the Cas12J guide RNA and / or Cas12J fusion protein is operably linked to a constitutive promoter.

[0280] The promoter can be a constitutively active promoter (i.e., a promoter that is constitutively in an active / “ON” state), it can be an inducible promoter (i.e., a promoter whose state (active / “ON” or inactive / “OFF”) is controlled by an external stimulus such as the presence of a particular temperature, compound, or protein), it can be a spatially restricted promoter (i.e., transcriptional control elements, enhancers, etc.) (e.g., a tissue-specific promoter, a cell type-specific promoter, etc.), and it can be a temporally restricted promoter (i.e., a promoter that is in an “ON” state or an “OFF” state during a particular stage of embryonic development or during a particular stage of a biological process (e.g., a hair follicle cycle in a mouse)).

[0281] Suitable promoters can be derived from viruses and can thus be referred to as viral promoters, or they can be derived from any organism, including prokaryotes or eukaryotes. Suitable promoters can be used to drive expression by any RNA polymerase (e.g., pol I, pol II, pol III). Exemplary promoters include, but are not limited to, the SV40 early promoter, the mouse mammary tumor virus long terminal repeat (LTR) promoter; the adenovirus major late promoter (Ad MLP); the herpes simplex virus (HSV) promoter, the cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMV IE), the Rous sarcoma virus (RSV) promoter, the human U6 small nuclear promoter (U6) (Miyagishi et al., Nature Biotechnology 20, 497-500 (2002)), the enhanced U6 promoter (e.g., Xia et al., Nucleic Acids Res. 2003 Sep 1; 31(17)), the human H1 promoter (H1), and the like.

[0282] In some cases, the nucleotide sequence encoding the Cas12J guide RNA is operably linked to (under the control of) a promoter that is operable in a eukaryotic cell (e.g., a U6 promoter, an enhanced U6 promoter, an H1 promoter, and the like). As will be appreciated by one of ordinary skill in the art, when a U6 promoter (e.g., in a eukaryotic cell) or another Pol III promoter is used to express an RNA (e.g., a guide RNA) from a nucleic acid (e.g., an expression vector), mutations can need to be made to the RNA if there are several T’s (which encode U in RNA) in a row. This is because a string of T’s (e.g., 5 T’s) in DNA can act as a terminator for polymerase III (Pol III). Thus, to ensure transcription of the guide RNA in a eukaryotic cell, it can sometimes be necessary to modify the sequence encoding the guide RNA to eliminate the effect of the T’s. In some cases, the nucleotide sequence encoding the Cas12J protein (e.g., a wild-type Cas12J protein, a nickase Cas12J protein, a dCas12J protein, a fusion Cas12J protein, and the like) is operably linked to a promoter that is operable in a eukaryotic cell (e.g., a CMV promoter, an EF1a promoter, an estrogen receptor-regulated promoter, and the like).

[0283] Examples of inducible promoters include, but are not limited to, a T7 RNA polymerase promoter, a T3 RNA polymerase promoter, an isopropyl-beta-D-thiogalactopyranoside (IPTG)-regulated promoter, a lactose-inducible promoter, a heat shock promoter, a tetracycline-regulated promoter, a steroid-regulated promoter, a metal-regulated promoter, an estrogen receptor-regulated promoter, and the like. Thus, an inducible promoter can be regulated by a molecule including, but not limited to, doxycycline; an estrogen and / or an estrogen analog; IPTG; and the like.

[0284] Examples of inducible promoters include, but are not limited to, chemically / biochemically regulated promoters and physically regulated promoters, such as alcohol-regulated promoters, tetracycline-regulated promoters (e.g., anhydrotetracycline (aTc)-responsive promoters and other tetracycline-responsive promoter systems, which include tetracycline repressor protein (tetR), tetracycline operator sequence (tetO), and tetracycline transactivator fusion protein (tTA)), steroid-regulated promoters (e.g., promoters based on rat glucocorticoid receptor, human estrogen receptor, moth ecdysone receptor, and promoters from the steroid / thyroid / retinoid receptor superfamily), metal-regulated promoters (e.g., promoters derived from metallothionein (a protein that binds and chelates metal ions) genes from yeast, mouse, and human), pathogenesis-regulated promoters (e.g., promoters induced by salicylic acid, ethylene, or benzothiadiazole (BTH)), temperature / heat-inducible promoters (e.g., heat shock promoters), and light-regulated promoters (e.g., light-responsive promoters from plant cells).

[0285] In some cases, the promoter is a spatially restricted promoter (i.e., a cell type-specific promoter, a tissue-specific promoter, etc.), such that in a multicellular organism, the promoter is active (i.e., “ON”) in a particular subset of cells. A spatially restricted promoter can also be referred to as an enhancer, a transcriptional control element, a control sequence, etc. Any convenient spatially restricted promoter can be used, so long as the promoter is functional in the target host cell (e.g., a eukaryotic cell; a prokaryotic cell).

[0286] In some cases, the promoter is a reversible promoter. Suitable reversible promoters, including reversible inducible promoters, are known in the art. Such reversible promoters can be isolated from and derived from a number of organisms, e.g., eukaryotes and prokaryotes. Modifications of reversible promoters derived from a first organism (e.g., a first prokaryote and a second eukaryote, a first eukaryote and a second prokaryote, etc.) for use in a second organism are well known in the art. Such reversible promoters and systems based on such reversible promoters but further comprising additional control proteins include, but are not limited to, alcohol-regulated promoters (e.g., alcohol dehydrogenase I (alcA) gene promoter, promoters responsive to alcohol trans-activator proteins (AlcR), etc.), tetracycline-regulated promoters (e.g., promoter systems including Tet activators, TetON, TetOFF, etc.), steroid-regulated promoters (e.g., rat glucocorticoid receptor promoter systems, human estrogen receptor promoter systems, retinoid promoter systems, thyroid promoter systems, ecdysone promoter systems, mifepristone promoter systems, etc.), metal-regulated promoters (e.g., metallothionein promoter systems, etc.), pathogenesis-associated regulated promoters (e.g., salicylic acid-regulated promoters, ethylene-regulated promoters, benzothiadiazole-regulated promoters, etc.), temperature-regulated promoters (e.g., heat shock inducible promoters (e.g., HSP-70, HSP-90, soybean heat shock promoters, etc.)), light-regulated promoters, synthetic inducible promoters, etc.

[0287] RNA polymerase III (Pol III) promoters can be used to drive expression of non-protein coding RNA molecules (e.g., guide RNAs). In some cases, a suitable promoter is a Pol III promoter. In some cases, a Pol III promoter is operably linked to a nucleotide sequence encoding a guide RNA (gRNA). In some cases, a Pol III promoter is operably linked to a nucleotide sequence encoding a single guide RNA (sgRNA). In some cases, a Pol III promoter is operably linked to a nucleotide sequence encoding a CRISPR RNA (crRNA). In some cases, a Pol III promoter is operably linked to a nucleotide sequence encoding a tracrRNA.

[0288] Non-limiting examples of Pol III promoters include U6 promoters, HI promoters, 5S promoters, adenovirus 2 (Ad2) VAI promoters, tRNA promoters, and 7SK promoters. See, e.g., Schramm and Hernandez (2002) Genes & Development 16:2593-2620. In some cases, the Pol III promoter is selected from the group consisting of a U6 promoter, a HI promoter, a 5S promoter, an adenovirus 2 (Ad2) VAI promoter, a tRNA promoter, and a 7SK promoter. In some cases, the nucleotide sequence encoding the guide RNA is operably linked to a promoter selected from the group consisting of a U6 promoter, a HI promoter, a 5S promoter, an adenovirus 2 (Ad2) VAI promoter, a tRNA promoter, and a 7SK promoter. In some cases, the nucleotide sequence encoding the single-stranded guide RNA is operably linked to a promoter selected from the group consisting of a U6 promoter, a HI promoter, a 5S promoter, an adenovirus 2 (Ad2) VAI promoter, a tRNA promoter, and a 7SK promoter.

[0289] Examples of promoters that can be used in conjunction with expression in plants, plant tissues, and plant cells described herein include, but are not limited to, the promoters described in U.S. Patent No. 6,437,217 (maize RS81 promoter), U.S. Patent No. 5,641,876 (rice actin promoter), U.S. Patent No. 6,426,446 (maize RS324 promoter), U.S. Patent No. 6,429,362 (maize PR-l promoter), U.S. Patent No. 6,232,526 (maize A3 promoter), U.S. Patent No. 6,177,611 (constitutive maize promoter), U.S. Patent Nos. 5,322,938, 5,352,605, 5,359,142, and 5,530,196 (35S promoter), U.S. Patent No. 6,433,252 (maize L3 oleosin promoter), U.S. Patent No. 6,429,357 (rice actin 2 promoter and rice actin 2 intron), U.S. Patent No. 5,837,848 (root-specific promoter), U.S. Patent No. 6,294,714 (light-inducible promoter), U.S. Patent No. 6,140,078 (salt-inducible promoter), U.S. Patent No. 6,252,138 (pathogen-inducible promoter), U.S. Patent No. 6,175,060 (phosphate-deficiency-inducible promoter), U.S. Patent No. 6,635,806 (gamma-zeatin promoter), and U.S. Patent Application No. 09 / 757,089 (maize chloroplast aldolase promoter).Additional promoters that can be used include the nopaline synthase (NOS) promoter (Ebert et al., 1987), the octopine synthase (OCS) promoter (carried on the tumor-inducing plasmid of Agrobacterium tumefaciens), a caulimovirus promoter such as the cauliflower mosaic virus (CaMV) 19S promoter (Lawton et al. Plant Molecular Biology (1987) 9:315-324), the CaMV 35S promoter (Odell et al., Nature (1985) 313:810-812), the figwort mosaic virus 35S-promoter (U.S. Patent No. 6,051,753; 5,378,619), the sucrose synthase promoter (Yang and Russell, Proceedings of the National Academy of Sciences, USA (1990) 87:4144-4148), the R gene complex promoter (Chandler et al., Plant Cell (1989) 1:1175-1183), and the chlorophyll a / b binding protein gene promoter PC1SV (U.S. Patent No. 5,850,019) and the AGRtu.nos (GenBank Accession No. V00087; Depicker et al., Journal of Molecular and Applied Genetics (1982) 1:561-573; Bevan et al., 1983) promoters.

[0290] Methods of introducing nucleic acids (e.g., nucleic acids comprising donor polynucleotide sequences, nucleic acids encoding one or more Cas12J proteins and / or Cas12J guide RNAs, etc.) into host cells are known in the art, and any convenient method can be used to introduce nucleic acids (e.g., expression constructs) into cells. Suitable methods include, for example, viral infection, transfection, lipofection, electroporation, calcium phosphate precipitation, polyethylenimine (PEI)-mediated transfection, DEAE-dextran-mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery, etc.

[0291] Introduction of the recombinant expression vector into a cell can occur in any medium that promotes cell survival and under any culture conditions. Introduction of the recombinant expression vector into a target cell can occur in vivo or ex vivo. Introduction of the recombinant expression vector into a target cell can occur in vitro.

[0292] In some embodiments, Cas12J proteins can be provided as RNA. RNA can be provided by direct chemical synthesis or can be transcribed in vitro from DNA (e.g., encoding a Cas12J protein). Once synthesized, RNA can be introduced into a cell by any of the well-known techniques for introducing nucleic acids into cells (e.g., microinjection, electroporation, transfection, etc.).

[0293] Well-developed transfection techniques can be used (see, e.g., Angel and Yanik (2010) PLoS ONE 5(7): el 1756); and the Lipofectamine® mRNA Transfection Kit, commercially available from Qiagen Stemfect, commercially available from Stemgent TM RNA Transfection Kit, and the Cell Line Nucleofector® Kit V, commercially available from Mirus Bio LLC The -mRNA Transfection Kit provides nucleic acids to cells. See also Beumer et al. (2008) PNAS 105(50): 19821-19826.

[0294] Vectors can be provided directly to target host cells. In other words, cells are contacted with a vector comprising a subject nucleic acid (e.g., a recombinant expression vector having a donor template sequence and encoding a Cas12J guide RNA; a recombinant expression vector encoding a Cas12J protein, etc.) such that the vector is taken up by the cell. Methods for contacting cells with nucleic acid vectors as plasmids, including electroporation, calcium chloride transfection, microinjection, and liposome transfection, are well known in the art. For viral vector delivery, cells can be contacted with viral particles comprising a subject viral expression vector.

[0295] Retroviruses, such as lentiviruses, are suitable for use in the methods of the disclosure. Typically used retroviral vectors are “defective,” i.e., incapable of producing the viral proteins required for productive infection. Moreover, replication of the vector requires growth in a packaging cell line. To generate viral particles comprising a nucleic acid of interest, a retroviral nucleic acid comprising the nucleic acid is packaged into a viral capsid by a packaging cell line. Different packaging cell lines provide different envelope proteins to be incorporated into the capsid (ecotropic, amphotropic, or xenotropic), which determine the specificity of the viral particles for cells (ecotropic for murine and rat; amphotropic for most mammalian cell types including human, dog, and mouse; and xenotropic for most mammalian cell types except murine cells). An appropriate packaging cell line can be used to ensure that cells are targeted by the packaged viral particles. Methods for introducing a subject vector expression vector into a packaging cell line and collecting viral particles generated by the packaging cell line are well known in the art. Nucleic acids can also be introduced by direct microinjection (e.g., injection of RNA).

[0296] A vector for providing a target host cell with a nucleic acid encoding a Casl2J guide RNA and / or Casl2J polypeptide can include a suitable promoter for driving expression (i.e., transcriptional activation) of the nucleic acid of interest. In other words, in some cases, the nucleic acid of interest will be operably linked to a promoter. The promoter can include a ubiquitous constitutive promoter, such as a CMV-beta-actin promoter; or an inducible promoter, such as a promoter active in a particular cell population or responsive to the presence of a drug, such as tetracycline. By transcriptional activation, it is contemplated that transcription will be increased 10-fold, 100-fold, more typically 1000-fold in the target cell compared to basal levels. Additionally, a vector for providing a cell with a nucleic acid encoding a Casl2J guide RNA and / or Casl2J protein can comprise a nucleic acid sequence that encodes a selectable marker in the target cell so as to identify cells that have taken up the Casl2J guide RNA and / or Casl2J protein.

[0297] A nucleic acid comprising a nucleotide sequence encoding a Casl2J polypeptide or Casl2J fusion polypeptide is in some cases RNA. Thus, a Casl2J fusion protein can be introduced into a cell in the form of RNA. Methods of introducing RNA into a cell are known in the art and can include, for example, direct injection, transfection, or any other method for introducing DNA. A Casl2J protein can alternatively be provided to a cell in the form of a polypeptide. Such a polypeptide can optionally be fused to a polypeptide domain that increases solubility of the product. The domain can be linked to the polypeptide by a defined protease cleavage site, such as a TEV sequence cleaved by a TEV protease. The linker can also include one or more flexible sequences, such as 1-10 glycine residues. In some embodiments, cleavage of the fusion protein is performed in a buffer that maintains solubility of the product, such as in the presence of 0.5-2 M urea, in the presence of a polypeptide and / or polynucleotide that increases solubility, etc. Target domains include endosomolysis domains, such as influenza HA domains; and other polypeptides that aid in production, such as IF2 domains, GST domains, GRPE domains, etc. The polypeptide can be formulated for improved stability. For example, the peptide can be PEGylated, where the polyethylenoxy provides increased lifetime in the bloodstream.

[0298] Additionally or alternatively, Cas12J polypeptides of the disclosure can be fused to a polypeptide penetration domain to facilitate uptake by a cell. Many penetration domains are known in the art and can be used with non-integrating polypeptides of the disclosure, including peptides, peptidomimetics, and non-peptide carriers. For example, a penetration peptide can be derived from the third alpha helix of the Drosophila melanogaster transcription factor Antennapedia gene, known as a penetratin, which comprises the amino acid sequence RQIKIWFQNR RMKWKK (SEQ ID NO: 68). As another example, a penetration peptide comprises the HIV-1 tat basic region amino acid sequence, which can include, for example, amino acids 49-57 of a naturally occurring tat protein. Other penetration domains include polyarginine motifs, such as the region of amino acids 34-56 of the HIV-1 rev protein, nona-arginine, octa-arginine, and the like. (See, e.g., Futaki et al. (2003) Curr Protein Pept Sci. 2003 Apr;4(2):87-9 and 446; and Wender et al. (2000) Proc. Natl. Acad. Sci. U.S.A 2000 Nov 21; 97(24): 13003-8; U.S. Patent Publication Nos. 20030220334; 20030083256; 20030032593; and 20030022831, the teachings of translocation peptides and peptoids are expressly incorporated by reference herein). The nona-arginine (R9) sequence is one of the more effective PTDs that have been characterized (Wender et al. 2000; Uemura et al. 2002). Sites for fusion can be selected in order to optimize the biological activity, secretion, or binding characteristics of the polypeptide. Optimal sites will be determined by routine experimentation.

[0299] As described above, in some cases, the target cell is a plant cell. Numerous methods of transforming chromosomes or plastids in plant cells with recombinant nucleic acids are known in the art and can be used in accordance with the methods of the present application to produce transgenic plant cells and / or transgenic plants. Any suitable method or technique known in the art for transforming plant cells can be used. Effective methods for transforming plants include bacterially-mediated transformation, such as Agrobacterium- or Rhizobium- mediated transformation and microprojectile bombardment-mediated transformation. A variety of methods are known in the art for transforming explants with a transformation vector via bacterially-mediated transformation or microprojectile bombardment and then subsequently culturing those explants to regenerate or develop transgenic plants, among others. Other methods for plant transformation are also known in the art, such as microinjection, electroporation, vacuum infiltration, pressure, sonication, silicon carbide fiber agitation, PEG-mediated transformation, and the like. Transgenic plants produced by these transformation methods can be chimeric or non-chimeric for the transformation event, depending on the method and explant used.

[0300] Methods for transforming plant cells are well known to those skilled in the art. For example, specific descriptions of transforming plant cells by microparticle bombardment with particles coated with recombinant DNA (e.g., bio-bomb transformation) are found in U.S. Patent Nos. 5,550,318; 5,538,880; 6,160,208; 6,399,861; and 6,153,812, and Agrobacterium-mediated transformation is described in U.S. Patent Nos. 5,159,135; 5,824,877; 5,591,616; 6,384,301; 5,750,871; 5,463,174; and 5,188,958. Other methods for transforming plants can be found, for example, in the Compendium of Transgenic Crop Plants (2009), Blackwell Publishing. Plant cells can be transformed with any nucleic acid provided herein using any suitable method known to those skilled in the art.

[0301] The Cas12J polypeptide disclosed herein can be produced in vitro or by eukaryotic or prokaryotic cells, and it can be further processed by unfolding (e.g., thermal denaturation, dithiothreitol reduction, etc.) and further refolded using methods known in the art.

[0302] Target modifications that do not alter the primary sequence include chemical derivatization of peptides, such as acylation, acetylation, carboxylation, and amidation. It also includes glycosylation modifications, such as those performed by altering the glycosylated form of the peptide during synthesis and processing or in further processing steps; and those performed by exposing the peptide to enzymes that affect glycosylation, such as mammalian glycosylation or deglycosylation enzymes. Sequences containing phosphorylated amino acid residues, such as phosphotyrosine, phosphotyserine, or phosphotythreonine, are also covered.

[0303] Also suitable to be included in embodiments of this disclosure are nucleic acids (e.g., nucleic acids encoding Cas12J guide RNA, Cas12J fusion proteins, etc.) and proteins (e.g., Cas12J fusion proteins derived from wild-type or variant proteins), which have been modified using common molecular biology techniques and synthetic chemistry to improve their resistance to proteolytic degradation, to alter target sequence specificity, to optimize solubility, to modify protein activity (e.g., transcriptional regulatory activity, enzyme activity, etc.), or to make them more suitable. Analogs of such peptides include those containing residues other than naturally occurring L-amino acids (e.g., D-amino acids or non-naturally occurring synthetic amino acids). The D-amino acids may replace some or all of the amino acid residues.

[0304] The Cas12J polypeptides of the disclosure can be prepared by in vitro synthesis using conventional methods as known in the art. Various commercial synthesis apparatuses, e.g., automated synthesizers of Applied Biosystems, Inc., Beckman, etc., can be used. By using the synthesizer, naturally occurring amino acids can be substituted with non-natural amino acids. The particular order and manner of preparation will be determined by convenience, economy, purity required, etc.

[0305] If desired, various groups can be introduced into the peptide during synthesis or during expression, which allow attachment to other molecules or surfaces. Thus, for example, cysteines can be used to make sulfides, histidines to attach to metal ion complexes, carboxyls to form amides or esters, aminos to form amides, etc.

[0306] The Cas12J polypeptides of the disclosure can also be isolated and purified according to conventional recombinant synthesis methods. A lysate can be prepared from the expression host, and the lysate purified using high performance liquid chromatography (HPLC), exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification techniques. In most cases, the composition used will constitute 20% or more by weight of the desired product, more usually 75% or more by weight, preferably 95% or more by weight, relative to contaminants associated with the method of production and purification thereof, and usually 99.5% or more by weight for therapeutic purposes. Typically, the percentage will be based on total protein. Thus, in some cases, the Cas12J polypeptides or Cas12J fusion polypeptides of the disclosure are at least 80% pure, at least 85% pure, at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure (e.g., free of contaminants, non-Cas12J proteins, or other macromolecules, etc.).

[0307] To induce cleavage or any desired modification of a target nucleic acid (e.g., genomic DNA), or any desired modification of a polypeptide associated with a target nucleic acid, a cell is provided with a Cas12J guide RNA and / or Cas12J polypeptide and / or donor template sequence of the disclosure (whether they are introduced as nucleic acids or polypeptides) for a period of about 30 minutes to about 24 hours, e.g., 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 16 hours, 18 hours, 20 hours, or any other period of time from about 30 minutes to about 24 hours, which can be repeated at a frequency of from about every day to about every 4 days, e.g., every 1.5 days, every 2 days, every 3 days, or any other frequency from about every day to about every four days. The subject cell(s) can be provided with one or more agents one or more times (e.g., once, twice, three times, or more than three times), and after each exposure event the cells are allowed to incubate with the agent(s) for an amount of time, e.g., 16-24 hours, after which the medium is replaced with fresh medium and the cells are further cultured.

[0308] In cases where two or more different targeting complexes are provided to a cell (e.g., two different Cas12J guide RNAs that are complementary to different sequences within the same or different target nucleic acids), the complexes can be provided (e.g., as two polypeptides and / or nucleic acids) or delivered simultaneously. Alternatively, the complexes can be provided sequentially, e.g., first providing a targeting complex, then providing a second targeting complex, etc., or vice versa.

[0309] To improve the delivery of DNA vectors to target cells, DNA can be protected from damage and facilitated into cells, e.g., by using lipoplexes and polyplexes. Thus, in some cases, a nucleic acid of the disclosure (e.g., a recombinant expression vector of the disclosure) can be covered with lipids in organized structures like micelles or liposomes. When the organized structure is complexed with DNA, it is called a lipoplex. There are three types of lipids, anionic lipids (negatively charged), neutral lipids, or cationic lipids (positively charged). Lipoplexes that utilize cationic lipids have been shown to be useful for gene transfer. Cationic lipids naturally complex with negatively charged DNA due to their positive charge. Additionally, due to their charge, they interact with cell membranes. Endocytosis of the lipoplex then occurs and the DNA is released into the cytoplasm. Cationic lipids also prevent degradation of the DNA by the cell.

[0310] Complexes of polymers with DNA are called polyplexes. Most polyplexes are composed of cationic polymers, and their production is regulated by ionic interactions. One major difference between the action methods of polyplexes and lipoplexes is that polyplexes cannot release their DNA payload into the cytoplasm, for which co-transfection with endosomolytic agents (agents that lyse endosomes, which are produced during endocytosis) such as inactivated adenoviruses must occur. However, this is not always the case; polymers such as polyethyleneimine have their own endosome-destroying methods, as do chitosan and trimethyl chitosan.

[0311] Dendrimers, a spherical, highly branched macromolecule, can also be used to genetically modify stem cells. The surface of a dendrimer particle can be functionalized to alter its properties. Specifically, cationic dendrimers (i.e., dendrimers with a positive surface charge) can be constructed. When genetic material such as a DNA plasmid is present, charge complementarity results in the transient association of the nucleic acid with the cationic dendrimer. The dendrimer-nucleic acid complex can be taken up into a cell by endocytosis when it reaches its destination.

[0312] In some cases, a nucleic acid of the present disclosure (e.g., an expression vector) comprises an insertion site for a guide sequence of interest. For example, a nucleic acid can comprise an insertion site for a guide sequence of interest, wherein the insertion site is immediately adjacent to a nucleotide sequence encoding a portion of a Cas12J guide RNA that does not change when the guide sequence changes to hybridize to a desired target sequence (e.g., a sequence that contributes to the Cas12J binding aspects of the guide RNA, e.g., a sequence that contributes to the dsRNA duplex of the Cas12J guide RNA - this portion of the guide RNA can also be referred to as the ‘scaffold’ or ‘constant region’ of the guide RNA). Thus, in some cases, a subject nucleic acid (e.g., an expression vector) comprises a nucleotide sequence encoding a Cas12J guide RNA, except that the portion encoding the guide sequence portion of the guide RNA is an insertion sequence (insertion site). An insertion site is any nucleotide sequence that is used for the insertion of a desired sequence. “Insertion sites” for various technologies are known to those of ordinary skill in the art, and any convenient insertion site can be used. An insertion site can be used for any method of manipulating a nucleic acid sequence. For example, in some cases, an insertion site is a multiple cloning site (MCS) (e.g., a site comprising one or more restriction enzyme recognition sequences), a site for cloning that is independent of ligation, a site for recombination-based cloning (e.g., recombination based on att sites), a nucleotide sequence recognized by a CRISPR / Cas (e.g., Cas9)-based technology, etc.

[0313] The insertion site can be any desired length, and can depend on the type of insertion site (e.g., can depend on whether the site contains one or more restriction enzyme recognition sequences (and how many), whether the site includes a target site for a CRISPR / Cas protein, etc.). In some cases, the insertion site of a subject nucleic acid is 3 or more nucleotides (nt) in length (e.g., 5 or more, 8 or more, 10 or more, 15 or more, 17 or more, 18 or more, 19 or more, 20 or more, or 25 or more, or 30 or more nt in length). In some cases, the insertion site of a subject nucleic acid is 2 to 50 nucleotides (nt) in length (e.g., 2 to 40 nt, 2 to 30 nt, 2 to 25 nt, 2 to 20 nt, 5 to 50 nt, 5 to 40 nt, 5 to 30 nt, 5 to 25 nt, 5 to 20 nt, 10 to 50 nt, 10 to 40 nt, 10 to 30 nt, 10 to 25 nt, 10 to 20 nt, 17 to 50 nt, 17 to 40 nt, 17 to 30 nt, 17 to 25 nt in length). In some cases, the insertion site of a subject nucleic acid is 5 to 40 nt in length.

[0314] [NUCLEIC ACID MODIFICATIONS]

[0315] In some embodiments, a subject nucleic acid (e.g., a Cas12J guide RNA) has one or more modifications (e.g., base modifications, backbone modifications, etc.) to provide the nucleic acid with a new or enhanced characteristic (e.g., improved stability). A nucleoside is a base-sugar combination. The base portion of the nucleoside is typically a heterocyclic ring. The two most common classes of heterocyclic rings are purines and pyrimidines. A nucleotide is a nucleoside that further includes a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to the 2', 3' or 5' hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link the nucleosides to one another to form a linear polymeric compound. In turn, the various ends of this linear polymeric compound can be further modified to form cyclic compounds, however, the linear compounds are suitable. In addition, the linear compounds can have internal nucleotide base complementarity and thus can fold upon themselves in a manner to produce a fully or partially double-stranded compound. Within oligonucleotide, the phosphate groups are commonly referred to as forming the intemucleoside backbone of the oligonucleotide. The normal bond or backbone of RNA and DNA is a 3' to 5' phosphodiester bond.

[0316] Suitable nucleic acid modifications include, but are not limited to, 2' O-methyl modified nucleotides, 2' fluoro-modified nucleotides, locked nucleic acid (LNA) modified nucleotides, peptide nucleic acid (PNA) modified nucleotides, nucleotides with phosphorothioate linkages, and 5' caps (e.g., 7-methylguanylate cap (m7G)). Additional details and additional modifications are described below.

[0317] 2'-O-methyl modified nucleotides (also known as 2'-O-methyl RNA) are a naturally occurring RNA modification found in tRNA and other small RNAs that occurs as a post-transcriptional modification. Oligonucleotides containing 2'-O-methyl RNA can be synthesized directly. This modification increases the Tm of RNA:RNA duplexes, but causes only minor changes in RNA:DNA stability. It is stable to attack by single-stranded ribonucleases and is typically 5-10 times less susceptible to DNAse than DNA. It is commonly used in antisense oligonucleotides as a means to increase stability and binding affinity to the target messenger.

[0318] 2' fluoro-modified nucleotides (e.g., 2' fluoro bases) have a fluorine-modified ribose that increases binding affinity (Tm) and also confers a degree of relative nuclease resistance compared to natural RNA. These modifications are commonly used in ribozymes and siRNA to improve stability in serum or other biological fluids.

[0319] LNA bases have a modification to the ribose backbone that locks the base in the C3'-internal position, which favors the RNA A-form duplex geometry. This modification significantly increases Tm and also has very strong nuclease resistance. Multiple LNAs can be inserted anywhere in an oligonucleotide except the 3' terminus. Applications have been described from antisense oligonucleotides to hybridization probes to SNP detection and allele-specific PCR. Because LNAs confer a large increase in Tm, they can also cause an increase in primer dimer formation and spontaneous hairpin formation. In some cases, the number of LNAs incorporated into a single oligonucleotide is 10 bases or less.

[0320] Phosphorothioate (PS) linkages (i.e., phosphorothioate linkages) replace the non-bridging oxygen in the phosphate backbone of a nucleic acid (e.g., an oligonucleotide) with a sulfur atom. This modification makes the internucleotide linkage resistant to nuclease degradation. Phosphorothioate linkages can be introduced between the last 3-5 nucleotides at the 5' or 3' terminus of an oligonucleotide to inhibit exonuclease degradation. Including phosphorothioate linkages within (e.g., throughout) an oligonucleotide can also help reduce endonuclease attack.

[0321] In some embodiments, a subject nucleic acid has one or more nucleotides that are 2'-0-methyl modified nucleotides. In some embodiments, a subject nucleic acid (e.g., dsRNA, siNA, etc.) has one or more 2' fluoro modified nucleotides. In some embodiments, a subject nucleic acid (e.g., dsRNA, siNA, etc.) has one or more LNA bases. In some embodiments, a subject nucleic acid (e.g., dsRNA, siNA, etc.) has one or more nucleotides linked by a phosphorothioate linkage (i.e., the subject nucleic acid has one or more phosphorothioate linkages). In some embodiments, a subject nucleic acid (e.g., dsRNA, siNA, etc.) has a 5' cap (e.g., a 7-methylguanylate cap (m7G)). In some embodiments, a subject nucleic acid (e.g., dsRNA, siNA, etc.) has a combination of modified nucleotides. For example, in addition to having one or more nucleotides with other modifications (e.g., 2'-0-methyl nucleotides and / or 2' fluoro modified nucleotides and / or LNA bases and / or phosphorothioate linkages), a subject nucleic acid (e.g., dsRNA, siNA, etc.) can also have a 5' cap (e.g., a 7-methylguanylate cap (m7G)).

[0322] [Modified backbones and modified internucleoside linkages]

[0323] Examples of suitable nucleic acids containing modifications (e.g., Cas12J guide RNAs) include nucleic acids containing modified backbones or non-natural internucleoside linkages. Nucleic acids with modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone.

[0324] Suitable modified oligonucleotide backbones containing a phosphorus atom include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates, 5'-alkylene phosphonates, and chiral phosphonates, phosphinates, phosphoramidates including 3 '-amino amino phosphoramidate and aminoalkylphosphoramidates, 2'-5' linked analogs of these, and those containing a normal 3'-5' link or alternatively a 2'-5' link between the last two nucleotides, as well as the analogs having reverse polarity wherein the adjacent linkages are 3' to 3', 5' to 5', or 2' to 2'. Suitable oligonucleotides having reverse polarity include those that comprise a single 3' to 3' linkage at the 3' terminal internucleotide linkage, i.e., a single inverted nucleoside residue which can be basic (nucleobaseless or replaced with a hydroxyl group). Also included are various salts (e.g., potassium or sodium), mixed salts, and free acid forms.

[0325] In some embodiments, the subject nucleic acid comprises one or more phosphorothioate and / or heteroatom internucleoside linkages, specifically -CH2-NH-O-CH2-, -CH2-N(CH3)-O-CH2- (referred to as a methylene(methylimino) or MMI backbone), -CH2-O-N(CH3)-CH2-, -CH2-N(CH3)-N(CH3)-CH2-, and -O-N(CH3)-CH2-CH2- (wherein the native phosphodiester nucleotide intemucleoside linkage is represented by -O-P(=O)(OH)-O-CH2-). MMI type internucleoside linkages are disclosed in U.S. Patent No. 5,489,677, the disclosure of which is incorporated by reference herein in its entirety. Suitable amide internucleoside linkages are disclosed in U.S. Patent No. 5,602,240, the disclosure of which is incorporated by reference herein in its entirety.

[0326] Also suitable are nucleic acids having morpholino backbone structures, as described, for example, in U.S. Patent No. 5,034,506. For example, in some embodiments, the subject nucleic acid comprises a 6-membered morpholino ring in place of the ribose ring. In some of these embodiments, a phosphorodiamidate or other non-phosphodiester internucleoside linkage replaces the phosphodiester linkage.

[0327] Suitable modified polynucleotide backbones that do not comprise a phosphorus atom have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatom non- or mixed N, O, and S internucleoside linkages. These include: those with heteroatom linkages, (formed entirely by oligonucleotide internucleoside linkages) having morpholino linkages (formed in part by the sugar portion of the nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2components.

[0328]

Mimics

[0329] The subject nucleic acid can be a nucleic acid mimic. The term "mimic" when applied to a polynucleotide is intended to encompass a polynucleotide in which only the furanose ring or both the furanose ring and the internucleotide linkage are replaced by non-furanose groups, the replacement of only the furanose ring also being referred to in the art as a sugar replacement. The heterocyclic base moiety or modified heterocyclic base moiety is maintained for hybridization with the appropriate target nucleic acid. One such nucleic acid, a polynucleotide mimic that has been shown to have superior hybridization properties, is a peptide nucleic acid (PNA). In a PNA, the sugar backbone of a polynucleotide is replaced with an amide-containing backbone, specifically an amino-ethylglycine backbone. The nucleotides are retained and are bonded directly or indirectly to the nitrogen heteroatoms of the amide moieties of the backbone.

[0330] One polynucleotide mimic that has been reported to have superior hybridization properties is a peptide nucleic acid (PNA). The backbone in a PNA compound is two or more linked amino-ethylglycine units that give the PNA an amide-containing backbone. The heterocyclic base moieties are bonded directly or indirectly to the nitrogen heteroatoms of the amide moieties of the backbone. Representative U.S. patents that describe the preparation of PNA compounds include, but are not limited to, U.S. Patent Nos. 5,539,082; 5,714,331; and 5,719,262, the disclosures of which are incorporated by reference herein in their entireties.

[0331] Another class of polynucleotide mimics that has been investigated is a linked morpholino unit (morpholino nucleic acid) based on a heterocyclic base having a morpholino ring attached. A number of linking groups for the morpholino monomer units in a linked morpholino nucleic acid have been reported. One class of linking groups has been selected to result in a non-ionic oligomeric compound. Non-ionic morpholino-based oligomeric compounds are less likely to have undesired interactions with cellular proteins. Morpholino-based polynucleotides are non-ionic mimics of oligonucleotides that are less likely to form undesired interactions with cellular proteins (Dwaine A. Braasch and David R. Corey, Biochemistry, 2002, 41(14), 4503-4510). Morpholino-based polynucleotides are disclosed in U.S. Patent No. 5,034,506, the disclosure of which is incorporated by reference herein in its entirety. A variety of compounds within the class of morpholino-based polynucleotides have been prepared, the compounds having a variety of different linking groups linking the monomer subunits.

[0332] Another class of polynucleotide mimics is referred to as cyclohexene-based nucleic acids (CeNA). The furanose ring normally present in DNA / RNA molecules is replaced by a cyclohexene ring. CeNA DMT-protected phosphoramidite monomers have been prepared and used for oligomeric compound synthesis according to classical phosphoramidite chemistry. Fully modified CeNA oligomeric compounds and oligonucleotides with specific positions modified with CeNA have been prepared and investigated (see Wang et al., J. Am. Chem. Soc, 2000, 122, 8595-8602, the disclosure of which is incorporated herein by reference in its entirety). Generally, incorporation of CeNA monomers into DNA strands increases the stability of DNA / RNA hybrids. CeNA oligoadenylates form complexes with RNA and DNA complementary sequences with similar stability to natural complexes. Studies showing the incorporation of CeNA structures into natural nucleic acid structures continue to make simple conformational adjustments by NMR and circular dichroism.

[0333] Another modification includes a locked nucleic acid (LNA), in which the 2'-hydroxyl group is linked to the 4' carbon atom of the sugar ring, thereby forming a 2'-C, 4'-C- oxy methylene linkage, thereby forming a bicyclic sugar moiety. The linkage can be a methylene (-CH2-), i.e., a group bridging the 2' oxygen atom and the 4' carbon atom, where n is 1 or 2 (Singh et al., Chem. Commun., 1998, 4, 455-456, the disclosure of which is incorporated herein by reference in its entirety). LNAs and LNA analogs exhibit very high duplex thermal stabilities (Tm = +3°C to +10°C) with complementary DNA and RNA, stability towards 3'-exonucleolytic degradation, and good solubility properties. Effective and non-toxic antisense oligonucleotides containing LNAs have been described (e.g., Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 5633-5638, the disclosure of which is incorporated herein by reference in its entirety).

[0334] The synthesis and preparation of LNA monomers adenine, cytosine, guanine, 5-methyl- cytosine, thymine, and uracil, along with their oligomerization and nucleic acid recognition properties, have been described (e.g., Koshkin et al., Tetrahedron, 1998, 54, 3607-3630, the disclosure of which is incorporated herein by reference in its entirety). LNAs and their preparation are also described in WO 98 / 39352 and WO 99 / 14226, as well as U.S. applications 20120165514, 20100216983, 20090041809, 20060117410, 20040014959, 20020094555, and 20020086998, the disclosures of which are incorporated herein by reference in their entireties.

[0335] [The modified sugar portion]

[0336] The subject nucleic acid may also contain one or more substituted sugar moieties. Suitable polynucleotides contain sugar substituents selected from the following: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-ynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl, and ynyl groups may be substituted or unsubstituted C1 to C2 groups. 10 Alkyl or C2 to C 10 Alkenyl and ynyl groups. Particularly suitable is: O((CH2) n O) m CH3, O(CH2) n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2 and O(CH2) n ON((CH2) n CH3)2, where n and m are 1 to approximately 10. Other suitable polynucleotides contain sugar substituents selected from the following: C1 to C2. 10 Lower alkyl groups, substituted lower alkyl groups, alkenyl groups, alkynyl groups, aryl groups, O-aryl or O-aryl groups, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocyclic alkyl groups, heterocyclic aryl groups, aminoalkylamino groups, polyalkylamino groups, substituted silyl groups, RNA cleaving groups, reporter groups, intercalating agents, groups that improve the pharmacokinetic properties of oligonucleotides, or groups that improve the pharmacodynamic properties of oligonucleotides, and other substituents with similar properties. Suitable modifications include 2'-methoxyethoxy (2'-O-CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504, the disclosure of which is incorporated herein by reference in its entirety), i.e. alkoxyalkoxy. Other suitable modifications include 2'-dimethylaminoethoxy, i.e., the O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, as described in the examples below; and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethyl-amino-ethoxy-ethyl or 2'-DMAEOE), i.e., 2'-O-CH2-O-CH2-N(CH3)2.

[0337] Other suitable sugar substituent groups include methoxy (-O-CH3), aminopropoxy (-O-CH2CH2CH2NH2), allyl (-CH2-CH=CH2), -O-allyl (-O-CH2-CH=CH2), and fluoro (F). The 2'-sugar substituent group can be in the arabinose (up) position or the ribose (down) position. A suitable 2'-arabinose modification is 2'-F. Similar modifications can also be made at other positions on the oligomeric compounds, particularly the 3' position of the sugar on the 3' terminal nucleoside or in 2'-5' linked oligonucleotides, and the 5' position of the 5' terminal nucleotide. Oligomeric compounds can also have sugar mimetics such as cyclobutyl moieties in place of furanose sugars.

[0338] [Base Modifications and Substitutions]

[0339] Subject nucleic acids can also include nucleobase (often simply referred to as "base" in the art) modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G) and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2- aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2- thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C=C-CH3) uracil and cytosine, and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo (ie 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine, 2-F-adenine, 2- amino-adenine, 8-azaguanine and 8-azadenine, 7-deazaguanine and 7-deazaadenine and 3- deazaguanine and 3-deazaadenine. Additional modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine (1H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamp such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido(5,4-(b)(1,4)benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido(4,5-b)indole-2-one), pyridoindole cytidine (H-pyrido(3',2':4,5)pyrrolo(2,3-d)pyrimidin-2-one).

[0340] Heterocyclic base moieties can also include those in which the purine or pyrimidine base is replaced by other heterocycles, such as 7-deazadenine, 7-deazaguanosine, 2- aminopyridine, and 2-pyridinone. Additional nucleobases include those disclosed in U.S. Patent No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J.I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S.T. and Lebleu, B., eds., CRC Press, 1993, the disclosures of which are incorporated herein by reference in their entirety. Certain of these nucleobases can be useful to increase the binding affinity of the oligomeric compounds. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6, and O-6 substituted purines, including 2- aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi et al. eds. Antisense Research and Applications, CRC Press, Boca Raton, 1993, pages 276-278; the disclosure of which is incorporated herein by reference in its entirety) and are suitable substitutions when combined with 2'-O-methoxyethyl sugar modifications, for example.

[0341]

Conjugates

[0342] Another possible modification of the subject nucleic acids involves chemically linking one or more moieties or conjugates to the polynucleotide that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide. These moieties or conjugates can include conjugate groups covalently bonded to functional groups such as primary or secondary hydroxyl groups. Conjugate groups include, but are not limited to, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Suitable conjugate groups include, but are not limited to, cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, include, but are not limited to, groups that improve uptake, enhance resistance to degradation, and / or strengthen sequence-specific hybridization to the target nucleic acid. Groups that enhance the pharmacokinetic properties include groups that improve uptake, distribution, metabolism or excretion of the subject nucleic acids.

[0343] Conjugate moieties include, but are not limited to, lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), a cholic acid moiety (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, for example, hexyl-S-tritylthiol (Manoharan et al., Ann. N. Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a

[0344] A conjugate can include a "protein transduction domain" or PTD (also known as a CPP - cell-penetrating peptide), which can refer to a polypeptide, polynucleotide, carbohydrate, or organic or inorganic compound that facilitates crossing of a lipid bilayer, micelle, cell membrane, organelle membrane, or vesicle membrane. A PTD attached to another molecule (which can range from a small polar molecule to a large macromolecule and / or nanoparticle) facilitates the molecule crossing a membrane, e.g., from an extracellular space into an intracellular space or from the cytosol into an organelle (e.g., nucleus). In some embodiments, a PTD is covalently linked to the 3' end of an exogenous polynucleotide. In some embodiments, a PTD is covalently linked to the 5' end of an exogenous polynucleotide. Exemplary PTDs include, but are not limited to, the minimal eleven-amino acid polypeptide protein transduction domain (corresponding to residues 47-57 of HIV-1 TAT comprising YGRKKRRQRRR; SEQ ID NO:64); a polyarginine sequence comprising a number of arginines sufficient to directly enter a cell (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10-50 arginines); a VP22 domain (Zender et al. (2002) Cancer Gene Ther. 9(6):489-96); a Drosophila Antennapedia gene protein transduction domain (Noguchi et al. (2003) Diabetes 52(7): 1732-1737); a truncated human calcitonin peptide (Trehin et al. (2004) Pharm. Research 21 : 1248-1256); a polylysine (Wender et al. (2000) Proc. Natl. Acad. Sci. USA 97: 13003-13008); RRQRRTSKLM KR (SEQ ID NO:65); a transportan protein GWTLNSAGYL LGKINLKALA ALAKKIL (SEQ ID NO:66); KALAWEAKLAKALAKALAKH LAKALAKALKCEA (SEQ ID NO:67); and RQIKIWFQNR RMKWKK (SEQ ID NO:68). Exemplary PTDs include, but are not limited to, YGRKKRRQRRR (SEQ ID NO:64), RKKRRQRRR (SEQ ID NO:69); an arginine homopolymer with 3 arginine residues to 50 arginine residues; exemplary PTD domain amino acid sequences include, but are not limited to, any one of the following: YGRKKRRQRRR (SEQ ID NO:64); RKKRRQRR (SEQ ID NO:69); YARAAARQAR A (SEQ ID NO:71); THRLPRRRRRR (SEQ ID NO:72); and GGRRARRRRRR (SEQ ID NO:73).In some embodiments, the PTD is an activatable CPP (ACPP) (Aguilera et al. (2009) Integr Biol (Camb) Jun; 1(5-6): 371-381). ACPPs include a polycationic CPP (e.g., Arg9 or "R9") linked via a cleavable linker to a matching polyanion (e.g., Glu9 or "E9"), which reduces the net charge to near zero and thereby inhibits adhesion and uptake into cells. When the linker is cleaved, the polyanion is released, locally exposing the polyarginine and its inherent adhesiveness, thereby "activating" the ACPP to cross the membrane.

[0345]

Introducing components into target cells

[0346] Cas12J guide RNAs (or nucleic acids comprising nucleotide sequences encoding Cas12J guide RNAs) and / or Cas12J polypeptides of the disclosure (or nucleic acids comprising nucleotide sequences encoding Cas12J polypeptides) and / or Cas12J fusion polypeptides of the disclosure (or nucleic acids comprising nucleotide sequences encoding Cas12J fusion polypeptides of the disclosure) and / or donor polynucleotides (donor templates) can be introduced into a host cell by any of a variety of well-known methods.

[0347] Any of a variety of compounds and methods can be used to deliver a Cas12J system of the disclosure to a target cell (e.g., wherein the Cas12J system comprises: (a) a Cas12J polypeptide of the disclosure and a Cas12J guide RNA; b) a Cas12J polypeptide of the disclosure, a Cas12J guide RNA, and a donor template nucleic acid; c) a Cas12J fusion polypeptide of the disclosure and a Cas12J guide RNA; d) a Cas12J fusion polypeptide of the disclosure, a Cas12J guide RNA, and a donor template nucleic acid; e) an mRNA encoding a Cas12J polypeptide of the disclosure; and a Cas12J guide RNA; f) an mRNA encoding a Cas12J polypeptide of the disclosure, a Cas12J guide RNA, and a donor template nucleic acid; g) an mRNA encoding a Cas12J fusion polypeptide of the disclosure; and a Cas12J guide RNA; h) an mRNA encoding a Cas12J fusion polypeptide of the disclosure, a Cas12J guide RNA, and a donor template nucleic acid; i) a recombinant expression vector comprising a nucleotide sequence encoding a Cas12J polypeptide of the disclosure and a nucleotide sequence encoding a Cas12J guide RNA; j) a recombinant expression vector comprising a nucleotide sequence encoding a Cas12J polypeptide of the disclosure, a nucleotide sequence encoding a Cas12J guide RNA, and a nucleotide sequence encoding a donor template nucleic acid; k) a recombinant expression vector comprising a nucleotide sequence encoding a Cas12J fusion polypeptide of the disclosure and a nucleotide sequence encoding a Cas12J guide RNA; l) a recombinant expression vector comprising a nucleotide sequence encoding a Cas12J fusion polypeptide of the disclosure, a nucleotide sequence encoding a Cas12J guide RNA, and a nucleotide sequence encoding a donor template nucleic acid; m) a first recombinant expression vector comprising a nucleotide sequence encoding a Cas12J polypeptide of the disclosure, and a second recombinant expression vector comprising a nucleotide sequence encoding a Cas12J guide RNA; n) a first recombinant expression vector comprising a nucleotide sequence encoding a Cas12J polypeptide of the disclosure, and a second recombinant expression vector comprising a nucleotide sequence encoding a Cas12J guide RNA; and a donor template nucleic acid; o) a first recombinant expression vector comprising a nucleotide sequence encoding a Cas12J fusion polypeptide of the disclosure, and a second recombinant expression vector comprising a nucleotide sequence encoding a Cas12J guide RNA; p) a first recombinant expression vector comprising a nucleotide sequence encoding a Cas12J fusion polypeptide of the disclosure, and a second recombinant expression vector comprising a nucleotide sequence encoding a Cas12J guide RNA; and a donor template nucleic acid; q) a recombinant expression vector comprising a nucleotide sequence encoding a Cas12J polypeptide of the disclosure, a nucleotide sequence encoding a first Cas12J guide RNA, and a nucleotide sequence encoding a second Cas12J guide RNA;or r) a recombinant expression vector comprising a nucleotide sequence encoding a Cas12J fusion polypeptide of the disclosure, a nucleotide sequence encoding a first Cas12J guide RNA, and a nucleotide sequence encoding a second Cas12J guide RNA; or a variant of any of (a) through (r). As one non-limiting example, a Cas12J system of the disclosure can be combined with a lipid. As another non-limiting example, a Cas12J system of the disclosure can be combined with, or formulated into, a particle.

[0348] Methods of introducing nucleic acids into host cells are known in the art, and any convenient method can be used to introduce a subject nucleic acid (e.g., expression construct / vector) into a target cell (e.g., prokaryotic cell, eukaryotic cell, plant cell, animal cell, mammalian cell, human cell, etc.). Suitable methods include, e.g., viral infection, transfection, conjugation, protoplast fusion, liposome transfection, electroporation, calcium phosphate precipitation, polyethylenimine (PEI)-mediated transfection, DEAE-dextran-mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery (see, e.g., Panyam et al. Adv Drug Deliv Rev. 2012 Sep 13. pii: S0169-409X(12)00283-9. doi: 10.1016 / j.addr.2012.09.023), and the like.

[0349] In some cases, a Cas12J polypeptide of the disclosure is provided as a nucleic acid (e.g., mRNA, DNA, plasmid, expression vector, viral vector, etc.) encoding the Cas12J polypeptide. In some cases, a Cas12J polypeptide of the disclosure is provided directly as a protein (e.g., not with an associated guide RNA or with an associated guide RNA, i.e., as a ribonucleoprotein complex). A Cas12J polypeptide of the disclosure can be introduced into a cell (provided to a cell) by any convenient method; such methods are known to those of ordinary skill in the art. As one illustrative example, a Cas12J polypeptide of the disclosure can be injected directly into a cell (e.g., with or without a Cas12J guide RNA or nucleic acid encoding a Cas12J guide RNA, and with or without a donor polynucleotide). As another example, a pre-formed complex (RNP) of a Cas12J polypeptide of the disclosure and a Cas12J guide RNA can be introduced into a cell (e.g., a eukaryotic cell) (e.g., via injection, via nucleofection; via conjugation to one or more components of a protein transduction domain (PTD), e.g., conjugation to a Cas12J protein, conjugation to a guide RNA, conjugation to a Cas12J polypeptide of the disclosure and a guide RNA of the disclosure; etc.).

[0350] In some cases, a Cas12J fusion polypeptide of the disclosure (e.g., a dCas12J fused to a fusion partner, a nickase Cas12J fused to a fusion partner, etc.) is provided as a nucleic acid (e.g., mRNA, DNA, plasmid, expression vector, viral vector, etc.) encoding the Cas12J fusion polypeptide. In some cases, a Cas12J fusion polypeptide of the disclosure is provided directly as a protein (e.g., not with or with an associated guide RNA, i.e., as a ribonucleoprotein complex). A Cas12J fusion polypeptide of the disclosure can be introduced into a cell (provided to a cell) by any convenient method; such methods are known to those of ordinary skill in the art. As one illustrative example, a Cas12J fusion polypeptide of the disclosure can be injected directly into a cell (e.g., with or without a nucleic acid encoding a Cas12J guide RNA, and with or without a donor polynucleotide). As another example, a preformed complex (RNP) of a Cas12J fusion polypeptide of the disclosure and a Cas12J guide RNA can be introduced into a cell (e.g., via injection, via nucleofection; via conjugation to a protein transduction domain (PTD) of one or more components, e.g., to a Cas12J fusion protein, to a guide RNA, to a Cas12J fusion polypeptide of the disclosure and a guide RNA; etc.).

[0351] In some cases, nucleic acids (e.g., Cas12J guide RNA; nucleic acids containing a nucleotide sequence encoding the Cas12J polypeptide of this disclosure; etc.) are delivered to cells (e.g., target host cells) in or associated with particles and / or polypeptides (e.g., Cas12J polypeptides; Cas12J fusion polypeptides). In some cases, the Cas12J system of this disclosure is delivered to cells in or associated with particles. The terms "particle" and "nanoparticle" are used interchangeably where appropriate. A recombinant expression vector containing the nucleotide sequence encoding the Cas12J polypeptide disclosed herein and / or the Cas12J guide RNA, an mRNA containing the nucleotide sequence encoding the Cas12J polypeptide disclosed herein, and the guide RNA may be delivered simultaneously using particles or a lipid coating; for example, the Cas12J polypeptide and the Cas12J guide RNA, as a complex (e.g., a ribonucleoprotein (RNP) complex), may be delivered via particles, such as delivery particles containing lipids or lipid-like substances and hydrophilic polymers (e.g., cationic lipids and hydrophilic polymers), for example, wherein the cationic lipid contains 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or 1,2-tetracosanoyl-sn-glycero-3-phosphocholine (DMPC) and / or wherein the hydrophilic polymer contains ethylene glycol or polyethylene glycol (PEG); and / or wherein the particles also contain cholesterol (e.g., particles from formulation 1 = DOTAP 100, DMPC 0, PEG). 0, Cholesterol 0; Formulation No. 2 = DOTAP 90, DMPC 0, PEG 10, Cholesterol 0; Formulation No. 3 = DOTAP 90, DMPC 0, PEG 5, Cholesterol 5). For example, a multi-step method can be used to form particles in which the Cas12J peptide and Cas12J guide RNA are mixed together, for example, in a 1:1 molar ratio, for example, at room temperature, for example, for example, for 30 minutes, for example, in sterile nuclease-free 1x phosphate-buffered saline (PBS); and the DOTAP, DMPC, PEG and cholesterol suitable for the formulation are dissolved separately in alcohol (e.g., 100% ethanol); and the two solutions are mixed together to form particles containing the complex.

[0352] The Cas12J polypeptides of the disclosure (or mRNA comprising a nucleotide sequence encoding a Cas12J polypeptide of the disclosure; or a recombinant expression vector comprising a nucleotide sequence encoding a Cas12J polypeptide of the disclosure) and / or Cas12J guide RNA (or nucleic acid, such as one or more expression vectors encoding a Cas12J guide RNA) can be delivered simultaneously using particles or lipid envelopes. For example, biodegradable core-shell structures with poly(beta-amino ester) (PBAE) cores encapsulated by a phospholipid bilayer shell can be used. In some cases, self-assembling bioadhesive polymer-based particles / nanoparticles are used; such particles / nanoparticles can be applied for oral delivery of peptides, intravenous delivery of peptides, and intranasal delivery of peptides, e.g., to the brain. Other embodiments are contemplated, such as oral absorption of hydrophobic drugs and ocular delivery. Molecular envelope technology can be used, which involves an engineered polymeric envelope that protects and delivers to the site of disease. Doses of about 5 mg / kg can be used, either in a single dose or in multiple doses, depending on various factors, e.g., the target tissue.

[0353] Lipidoid compounds (e.g., as described in U.S. Patent Application 20110293703) can also be used to administer a polynucleotide, and can be used to deliver a Cas12J polypeptide of the disclosure, a Cas12J fusion polypeptide of the disclosure, an RNP of the disclosure, a nucleic acid of the disclosure, or a Cas12J system of the disclosure (e.g., wherein the Cas12J system comprises: (a) a Cas12J polypeptide of the disclosure and a Cas12J guide RNA; b) a Cas12J polypeptide of the disclosure, a Cas12J guide RNA, and a donor template nucleic acid; c) a Cas12J fusion polypeptide of the disclosure and a Cas12J guide RNA; d) a Cas12J fusion polypeptide of the disclosure, a Cas12J guide RNA, and a donor template nucleic acid; e) an mRNA encoding a Cas12J polypeptide of the disclosure; and a Cas12J guide RNA; f) an mRNA encoding a Cas12J polypeptide of the disclosure, a Cas12J guide RNA, and a donor template nucleic acid; g) an mRNA encoding a Cas12J fusion polypeptide of the disclosure; and a Cas12J guide RNA; h) an mRNA encoding a Cas12J fusion polypeptide of the disclosure, a Cas12J guide RNA, and a donor template nucleic acid; i) a recombinant expression vector comprising a nucleotide sequence encoding a Cas12J polypeptide of the disclosure and a nucleotide sequence encoding a Cas12J guide RNA; j) a recombinant expression vector comprising a nucleotide sequence encoding a Cas12J polypeptide of the disclosure, a nucleotide sequence encoding a Cas12J guide RNA, and a nucleotide sequence encoding a donor template nucleic acid; k) a recombinant expression vector comprising a nucleotide sequence encoding a Cas12J fusion polypeptide of the disclosure and a nucleotide sequence encoding a Cas12J guide RNA; l) a recombinant expression vector comprising a nucleotide sequence encoding a Cas12J fusion polypeptide of the disclosure, a nucleotide sequence encoding a Cas12J guide RNA, and a nucleotide sequence encoding a donor template nucleic acid; m) a first recombinant expression vector comprising a nucleotide sequence encoding a Cas12J polypeptide of the disclosure, and a second recombinant expression vector comprising a nucleotide sequence encoding a Cas12J guide RNA; n) a first recombinant expression vector comprising a nucleotide sequence encoding a Cas12J polypeptide of the disclosure, and a second recombinant expression vector comprising a nucleotide sequence encoding a Cas12J guide RNA; and a donor template nucleic acid; o) a first recombinant expression vector comprising a nucleotide sequence encoding a Cas12J fusion polypeptide of the disclosure, and a second recombinant expression vector comprising a nucleotide sequence encoding a Cas12J guide RNA; p) a first recombinant expression vector comprising a nucleotide sequence encod...

Claims

1. A composition comprising: (a) A polypeptide or a nucleic acid molecule encoding the polypeptide, wherein the polypeptide comprises the following amino acid sequence: (i) The amino acid sequence shown in any one of SEQ ID NO: 109, 110, 112, 120 and 126, or (ii) A polypeptide containing a mutated amino acid sequence in the RuvC domain, said mutation resulting in a polypeptide having reduced catalytic activity compared to a polypeptide composed of amino acid sequences having 100% identity with any one of SEQ ID NO: 109, 110, 112, 120, and 126, wherein (1) The amino acid sequence comprises any one of the amino acid sequences shown in SEQ ID NO: 109, 110, 112, 120, and 126, which have one, two, or three mutations at positions corresponding to D464, E678, and D769 of SEQ ID NO: 113, and (2) The mutation is a substitution of alanine (A); and (b) a guide RNA or one or more DNA molecules encoding the guide RNA, wherein the guide RNA comprises: (i) Nucleotide sequences complementary to the target sequence, and (ii) The region that binds to the polypeptide. The polypeptide and the guide RNA form a ribonucleoprotein complex that targets the target sequence through base pairing between the guide RNA and the target sequence.

2. The composition of claim 1, wherein the region of the guide RNA that binds to the polypeptide consists of a nucleotide sequence represented by any one of SEQ ID NO: 177, 178, 179 and 181.

3. The composition of claim 1, wherein the polypeptide is fused with a nuclear localization signal (NLS).

4. The composition according to any one of claims 1 to 3, further comprising a DNA donor template.

5. A fusion polypeptide composed of polypeptides fused with a heteropeptide, wherein... The polypeptide consists of the following amino acid sequence: (i) The amino acid sequence shown in any one of SEQ ID NO: 109, 110, 112, 120 and 126, or (ii) A polypeptide containing a mutated amino acid sequence in the RuvC domain, said mutation resulting in a polypeptide having reduced catalytic activity compared to a polypeptide composed of amino acid sequences having 100% identity with any one of SEQ ID NO: 109, 110, 112, 120, and 126, wherein (1) The amino acid sequence comprises any one of the amino acid sequences shown in SEQ ID NO: 109, 110, 112, 120, and 126, which have one, two, or three mutations at positions corresponding to D464, E678, and D769 of SEQ ID NO: 113, and (2) The mutation is a substitution of alanine (A); and The polypeptide binds to the guide RNA, and through base pairing between the guide RNA and the target sequence, a ribonucleoprotein complex targeting the target sequence is formed.

6. The fusion polypeptide of claim 5, wherein the fusion polypeptide is fused with a nuclear localization signal (NLS).

7. A composition containing one or more nucleic acids, wherein the composition contains: (a) A nucleotide sequence encoding a guide RNA, wherein the guide RNA comprises: (i) Nucleotide sequences complementary to the target sequence, and (ii) the region that binds to the polypeptide, and (b) The nucleotide sequence encoding the polypeptide, the polypeptide comprising the following amino acid sequence: (i) The amino acid sequence shown in any one of SEQ ID NO: 109, 110, 112, 120 and 126, or (ii) A polypeptide containing a mutated amino acid sequence in the RuvC domain, said mutation resulting in a polypeptide having reduced catalytic activity compared to a polypeptide composed of amino acid sequences having 100% identity with any one of SEQ ID NO: 109, 110, 112, 120, and 126, wherein (1) The amino acid sequence comprises any one of the amino acid sequences shown in SEQ ID NO: 109, 110, 112, 120, and 126, which have one, two, or three mutations at positions corresponding to D464, E678, and D769 of SEQ ID NO: 113, and (2) The mutation is a substitution of alanine (A); and The polypeptide and the guide RNA form a ribonucleoprotein complex that targets the target sequence through base pairing between the guide RNA and the target sequence.

8. The composition of claim 7, wherein the polypeptide is fused with a nuclear localization signal (NLS).

9. The composition of claim 7 or 8, wherein the one or more nucleic acids are one or more recombinant expression vectors, and the one or more recombinant expression vectors are selected from: one or more adeno-associated viral vectors, one or more recombinant retroviral vectors, or one or more recombinant lentiviral vectors.

10. Eukaryotic cells containing one or more of the following: (a) to (c) (a) A first polypeptide or a nucleic acid containing a nucleotide sequence encoding the first polypeptide, wherein the first polypeptide comprises the following amino acid sequence: (i) The amino acid sequence shown in any one of SEQ ID NO: 109, 110, 112, 120 and 126, or (ii) A polypeptide containing a mutated amino acid sequence in the RuvC domain, said mutation resulting in a polypeptide having reduced catalytic activity compared to a polypeptide composed of amino acid sequences having 100% identity with any one of SEQ ID NO: 109, 110, 112, 120, and 126, wherein (1) The amino acid sequence comprises any one of the amino acid sequences shown in SEQ ID NO: 109, 110, 112, 120, and 126, which have one, two, or three mutations at positions corresponding to D464, E678, and D769 of SEQ ID NO: 113, and (2) The mutation is a substitution of alanine (A). (b) A fusion polypeptide consisting of a first polypeptide fused with a heteropeptide, or a nucleic acid containing a nucleotide sequence encoding the fusion polypeptide, and (c) a guide RNA or a nucleic acid containing a nucleotide sequence encoding the guide RNA, wherein the guide RNA contains: (i) Nucleotide sequences complementary to the target sequence, and (ii) The region that binds to the first polypeptide or the fusion polypeptide. The first polypeptide or fusion polypeptide and the guide RNA form a ribonucleoprotein complex targeting the target sequence through base pairing between the guide RNA and the target sequence. The eukaryotic cells mentioned are not human germ cells, and The eukaryotic cells mentioned are not plant cells.

11. The eukaryotic cell of claim 10, comprising the nucleic acid described in (a), (b) or (c), wherein the nucleic acid is integrated into the genomic DNA of the cell.

12. The eukaryotic cell of claim 10 or claim 11, wherein the cell is a mammalian cell or an invertebrate cell.

13. The eukaryotic cell of claim 10 or claim 11, wherein the cell is an insect cell, a spider cell, a fungal cell, a bird cell, a reptile cell, an amphibian cell, a mouse cell, a rat cell, a non-human primate cell, or a human cell.

14. Cells, which contain: The fusion polypeptide of claim 5 or 6, or Nucleic acid containing the nucleotide sequence encoding the fusion polypeptide, The cells described therein are not human germ cells, and The cells mentioned are not plant cells.

15. The cell of claim 14, wherein it is a prokaryotic cell.

16. The cell of claim 14, comprising: the nucleic acid containing the nucleotide sequence encoding the fusion polypeptide, the nucleic acid being integrated into the genomic DNA of the cell.

17. The cell of claim 15, comprising: the nucleic acid containing the nucleotide sequence encoding the fusion polypeptide, the nucleic acid being integrated into the genomic DNA of the cell.

18. The cell according to any one of claims 15 to 17, comprising guide RNA, said guide RNA comprising: (i) Nucleotide sequences complementary to the target sequence, and (ii) The region that binds to the fusion polypeptide. The fusion polypeptide and the guide RNA form a ribonucleoprotein complex that targets the target sequence through base pairing between the guide RNA and the target sequence.

19. An in vitro or ex vivo method for modifying a target nucleic acid, wherein the method comprises: Contact the target nucleic acid with the following substances: (a) A polypeptide comprising the amino acid sequence shown in any one of SEQ ID NO: 109, 110, 112, 120, and 126, and (b) Guide RNA, which contains: (i) Nucleotide sequences complementary to the target sequence, and (ii) The region that binds to the polypeptide. The polypeptide and the guide RNA form a ribonucleoprotein complex that targets the target sequence through base pairing between the guide RNA and the target sequence. The contact results in the target nucleic acid being modified by the polypeptide, and The target nucleic acid is not found in human reproductive cells.

20. The in vitro or ex vivo method of claim 19, wherein the modification is the cleavage of the target nucleic acid.

21. The in vitro or ex vivo method of claim 19 or claim 20, wherein the target nucleic acid is selected from: double-stranded DNA, single-stranded DNA, RNA, genomic DNA, and extrachromosomal DNA.

22. In vitro or ex vivo methods for regulating transcription of a target nucleic acid, modifying a target nucleic acid, or modifying a protein that binds to a target nucleic acid, including: Contact the target nucleic acid with the following substances: (a) A fusion polypeptide consisting of a first polypeptide fused with a heteropeptide, wherein the first polypeptide comprises the following amino acid sequence: (i) The amino acid sequence shown in any one of SEQ ID NO: 109, 110, 112, 120 and 126, or (ii) A polypeptide containing a mutated amino acid sequence in the RuvC domain, said mutation resulting in a polypeptide having reduced catalytic activity compared to a polypeptide composed of amino acid sequences having 100% identity with any one of SEQ ID NO: 109, 110, 112, 120, and 126, wherein (1) The amino acid sequence comprises any one of the amino acid sequences shown in SEQ ID NO: 109, 110, 112, 120, and 126, which have one, two, or three mutations at positions corresponding to D464, E678, and D769 of SEQ ID NO: 113, and (2) The mutation is a substitution of alanine (A); and (b) Guide RNA, which contains: (i) Nucleotide sequences complementary to the target sequence, and (ii) the region that binds to the first polypeptide. The first polypeptide and the guide RNA form a ribonucleoprotein complex targeting the target sequence through base pairing between the guide RNA and the target sequence, and The target nucleic acid is not found in human reproductive cells.

23. The in vitro or ex vivo method of claim 22, wherein the fusion polypeptide is fused with a nuclear localization signal.

24. A method for producing a transgenic multicellular nonhuman organism, comprising introducing a transgene containing a nucleotide sequence into the genome of said nonhuman organism, said nucleotide sequence encoding one or more of the following: (a) A polypeptide consisting of the following amino acid sequence: (i) The amino acid sequence shown in any one of SEQ ID NO: 109, 110, 112, 120 and 126, or (ii) A polypeptide containing a mutated amino acid sequence in the RuvC domain, said mutation resulting in a polypeptide having reduced catalytic activity compared to a polypeptide composed of amino acid sequences having 100% identity with any one of SEQ ID NO: 109, 110, 112, 120, and 126, wherein (1) The amino acid sequence comprises any one of the amino acid sequences shown in SEQ ID NO: 109, 110, 112, 120, and 126, which have one, two, or three mutations at positions corresponding to D464, E678, and D769 of SEQ ID NO: 113, and (2) The mutation is a substitution of alanine (A). (b) the fusion polypeptide of claim 5 or 6, and (c) Guide RNA, which contains: (i) Nucleotide sequences complementary to the target sequence, and (ii) The region that binds to the polypeptide. The polypeptide and the guide RNA form a ribonucleoprotein complex that targets the target sequence through base pairing between the guide RNA and the target sequence.

25. The method of claim 24, wherein the organism is: a plant, an invertebrate, or a vertebrate.

26. The method of claim 24, wherein the organism is: a monocotyledonous plant, a dicotyledonous plant, an insect, an arthropod, an arachnid, a parasite, a worm, a cnidarian, a fish, a reptile, an amphibian, an ungulate, a bird, a pig, a horse, a sheep, a rodent, a mouse, a rat, or a non-human primate.

27. A kit containing one of the following (a) to (l): (a) First polypeptide and guide RNA, (b) First polypeptide, guide RNA and DNA donor template, (c) A fusion polypeptide consisting of a first polypeptide fused with a heteropeptide and guide RNA, (d) A fusion polypeptide consisting of a first polypeptide fused with a heteropeptide, a guide RNA and a DNA donor template, (e) mRNA encoding the first polypeptide and its guide RNA, (f) mRNA encoding the first polypeptide, guide RNA, and DNA donor template. (g) The mRNA and guide RNA encoding the fusion polypeptide consisting of the first polypeptide fused with the heteropeptide, (h) The mRNA, guide RNA, and DNA donor template encoding the fusion polypeptide consisting of the first polypeptide fused with the heteropeptide. (i) One or more recombinant expression vectors, including: (i) the nucleotide sequence encoding the first polypeptide, and (ii) The nucleotide sequence encoding the guide RNA, (j) One or more recombinant expression vectors, including: (i) The nucleotide sequence encoding the first polypeptide, (ii) The nucleotide sequence encoding the guide RNA, and (iii) DNA donor template, (k) One or more recombinant expression vectors, including: (i) The nucleotide sequence encoding the fusion polypeptide consisting of the first polypeptide fused with the heteropeptide, and (ii) the nucleotide sequence encoding the guide RNA, and (l) One or more recombinant expression vectors, including: (i) The nucleotide sequence encoding the fusion polypeptide consisting of a first polypeptide fused with a heteropeptide, and (ii) The nucleotide sequence encoding the guide RNA and the DNA donor template. The first polypeptide consists of an amino acid sequence represented by any one of SEQ ID NO: 109, 110, 112, 120, and 126. The guide RNA contains: (i) Nucleotide sequences complementary to the target sequence, and (ii) the region that binds to the first polypeptide, and The first polypeptide and the guide RNA form a ribonucleoprotein complex that targets the target sequence through base pairing between the guide RNA and the target sequence.

28. The kit of claim 27, wherein the donor template nucleic acid has a length of 8 to 1000 nucleotides.

29. The kit of claim 27, wherein the donor template nucleic acid has a length of 25 to 500 nucleotides.

30. A method for detecting target DNA in a sample, comprising: (a) Contact the sample with the following substances: (i) A polypeptide consisting of any one of the amino acid sequences shown in SEQ ID NO: 109, 110, 112, 120, and 126. (ii) Guide RNA, which contains: [a] Nucleotide sequences complementary to the target sequence, and [b]The region where the polypeptide binds, and (iii) Detector DNA, which is single-stranded and does not hybridize with the guide RNA. This allows the polypeptide and guide RNA to form a ribonucleoprotein complex targeting the target sequence through base pairing between the guide RNA and the target sequence. (b) The target DNA is detected by measuring a detectable signal generated by the cleavage of the single-stranded detector DNA of the polypeptide.

Images