CASZ compositions and methods of use

Abstract

Compositions and methods are provided that include one or more of the following: (1) a "CasZ" protein (also referred to as a CasZ polypeptide), a nucleic acid encoding the CasZ protein, and / or a modified host cell comprising the CasZ protein (and / or a nucleic acid encoding the CasZ protein); (2) a CasZ guide RNA that binds to the CasZ protein and provides sequence specificity for the CasZ protein, a nucleic acid encoding the CasZ guide RNA, and / or a modified host cell comprising the CasZ guide RNA (and / or a nucleic acid encoding the CasZ guide RNA); and (3) a CasZ trans-activating noncoding RNA (trancRNA) (referred to herein as "CasZ trancRNA"), a nucleic acid encoding the CasZ trancRNA, and / or a modified host cell comprising the CasZ trancRNA (and / or a nucleic acid encoding the CasZ trancRNA).

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 580,395, filed November 1, 2017, which is incorporated herein by reference in its entirety.

[0003] Incorporation by reference into the sequence listing provided as a text file

[0004] A sequence listing is hereby provided as the text file "BERK-374WO_SEQ_LISTING_ST25.txt", which was created on October 30, 2018 and is 536KB in size. The contents of the text file are incorporated herein by reference in their entirety.

[0005] introduction

[0006] The CRISPR-Cas system is an example of a path unknown to science before the era of DNA sequencing, and is now thought to confer acquired immunity to bacteriophages and viruses on bacteria and archaea. In-depth studies have revealed the biochemistry of this system. The CRISPR-Cas system consists of Cas proteins and CRISPR arrays, which are involved in the acquisition, targeting and cutting of exogenous DNA or RNA, and the CRISPR array includes direct repeats of short spacer sequences flanking the Cas proteins to their targets. Class 2 CRISPR-Cas is a streamlined version in which a single Cas protein bound to RNA is responsible for binding and cutting the targeting sequence. The programmable nature of these minimal systems facilitates their use as a versatile technology that is revolutionizing the field of genome manipulation. Summary of the invention

[0007] The present disclosure provides compositions and methods comprising one or more of the following: (1) a "CasZ" protein (also referred to as a CasZ polypeptide), a nucleic acid encoding a CasZ protein, and / or a modified host cell comprising a CasZ protein (and / or a nucleic acid encoding the CasZ protein); (2) a CasZ guide RNA that binds to a CasZ protein and provides sequence specificity for the CasZ protein, a nucleic acid encoding a CasZ guide RNA, and / or a modified host cell comprising a CasZ guide RNA (and / or a nucleic acid encoding the CasZ guide RNA); and (3) a CasZ trans-activating noncoding RNA (trancRNA) (referred to herein as "CasZtrancRNA"), a nucleic acid encoding a CasZtrancRNA, and / or a modified host cell comprising a CasZtrancRNA (and / or a nucleic acid encoding the CasZtrancRNA). BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 Examples of naturally occurring CasZ protein sequences are depicted.

[0009] Figure 2 A schematic diagram depicting the CasZ locus, which includes the Cas1 protein in addition to the CasZ protein.

[0010] Figure 3 Phylogenetic tree depicting CasZ sequences in relation to the other two classes of CRISPR / Cas effector protein sequences.

[0011] Figure 4 Phylogenetic tree depicting Cas1 sequences from the CasZ locus in relation to Cas1 sequences from the other 2 classes of CRISPR / Cas loci.

[0012] Figure 5 Describe transcriptomics RNA mapping data, which demonstrates the expression of trancRNA from the CasZ locus.trancRNA is adjacent to the CasZ repeat array, but does not contain repeats and is not complementary to repeats.Shown are RNA mapping data for the following loci: CasZa3, CasZb4, CasZc5, CasZd1, and CasZe3.Small repeat alignment arrows represent the repeats of the CRISPR array (indicating the presence of sequences encoding guide RNAs); peaks outside and near the repeat array represent highly transcribed trancRNAs. Figure 5 (Continued 1) Nucleotide sequence (from top to bottom: SEQ ID NO.: 312-331). Figure 5 (Continued 3) Nucleotide sequence (from top to bottom: SEQ ID No.: 161-177).

[0013] Figure 6 Depicted are the results of PAM preference for CasZc (top) and CasZb (bottom) as determined using a PAM depletion assay.

[0014] Figure 7 Depicts the sequence of the Cas14 protein described herein.

[0015] Figure 8 , Panels A-D depict the architecture and phylogeny of the CRISPR-Cas14 genomic locus.

[0016] Fig. 9 Depicting the phylogenetic analysis of Cas14 orthologs.

[0017] Fig.10 Depicting a maximum likelihood tree of Cas1 from known CRISPR systems.

[0018] Figure 11, panels A-B depict the acquisition of new spacer sequences by the CRISPR-Cas14 system.

[0019] Figure 12, Panels A-D depict that CRISPR-Cas14a actively adapts and encodes tracrRNA.

[0020] Fig.13 , Panels A-B depict the metatranscriptomics of the CRISPR-Cas14 locus.

[0021] FIG. 14 , Panels A-B depict RNA processing and heterologous expression of CRISPR-Cas14.

[0022] Fig.15 , Panels A-D depict plasmid depletion of Cas14a1 and SpCas9.

[0023] Fig.16 , Panels A-D depict that CRISPR-Cas14a is an RNA-guided DNA endonuclease.

[0024] Fig.17 , Figures A-E depict the degradation of ssDNA by Cas14a1.

[0025] Fig.18 Depicting the kinetics of ssDNA cleavage by Cas14a1 under various guide RNA compositions.

[0026] Fig.19 , Panels A-F depict optimization of Cas14a1 guide RNA components.

[0027] Fig. 20 , Figures AE depict high-fidelity ssDNA DNP detection by CRISPR-Cas14a. Fig. 20 , Figure C provides the nucleotide sequence (from top to bottom: SEQ ID NO: 367-370).

[0028] Fig.21 , Panels A-F depict the effects of various activators on the cleavage rate of Cas14a1.

[0029] Fig. 22 , Panels A-B depict the diversity of the CRISPR-Cas14 system.

[0030] Fig.23 , Panels A-C depict testing of Cas14a1-mediated interference in a heterologous host. Diagram of Cas14a1 and LbCas12a constructs used to test interference in E. coli.

[0031] Fig.24 Depicts the Cas14 nucleotide sequences of the plasmids used in the present invention.

[0032] Fig.25 , Figures A-E depict Fig.24 Sequence maps of each plasmid published in .

[0033] definition

[0034] As used herein, "heterologous" means a nucleotide or polypeptide sequence that is not present in a natural nucleic acid or protein, respectively. For example, relative to a CasZ polypeptide, a heterologous polypeptide comprises an amino acid sequence from a protein other than a CasZ polypeptide. In some cases, a portion of a CasZ protein from one species is fused with a portion of a CasZ protein from a different species. Therefore, it can be considered that the CasZ sequences from each species are heterologous relative to each other. As another example, a CasZ protein (e.g., a dCasZ protein) can be fused with an active domain from a non-CasZ protein (e.g., a histone deacetylase), and the sequence of the active domain can be considered to be a heterologous polypeptide (it is heterologous to the CasZ protein).

[0035] The terms "polynucleotide" and "nucleic acid" used interchangeably herein refer to a polymeric form of nucleotides (ribonucleotides or deoxynucleotides) of any length. Thus, this term includes, but is not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine bases and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derived nucleotide bases. The terms "polynucleotide" and "nucleic acid" should be understood to include single-stranded (such as sense or antisense strands) and double-stranded polynucleotides as applicable to the described embodiments.

[0036] The terms "polypeptide", "peptide" and "protein" used interchangeably herein refer to a polymeric form of amino acids of any length, which may include genetically encoded and non-genetically encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones. The terms include: fusion proteins, including but not limited to fusion proteins with heterologous amino acid sequences; fusions with heterologous and homologous leader sequences, with or without N-terminal methionine residues; immunolabeled proteins; etc.

[0037] As used herein, the term "naturally-occurring" as applied to a nucleic acid, protein, cell, or organism refers to a nucleic acid, cell, protein, or organism that occurs in nature.

[0038] As used herein, the term "isolated" is intended to describe a polynucleotide, polypeptide or cell that is in an environment different from that in which the polynucleotide, polypeptide or cell naturally occurs. An isolated genetically modified host cell may be present in a mixed population of genetically modified host cells.

[0039] As used herein, the term "exogenous nucleic acid" refers to nucleic acids that are not normally or naturally occurring in nature and / or are not produced by a given bacterium, organism, or cell. As used herein, the term "endogenous nucleic acid" refers to nucleic acids that are normally occurring in nature and / or are produced by a given bacterium, organism, or cell. "Endogenous nucleic acid" is also referred to as "native nucleic acid" or nucleic acids that are "native" to a given bacterium, organism, or cell.

[0040] As used herein, "recombinant" means that a particular nucleic acid (DNA or RNA) is the product of various combinations of cloning, restriction and / or ligation steps, which produce a construct having a structural coding sequence or non-coding sequence that can be distinguished from an endogenous nucleic acid present in a natural system. Generally speaking, the DNA sequence encoding the structural coding sequence can be assembled by a cDNA fragment and a short oligonucleotide linker or by a series of synthetic oligonucleotides to provide a synthetic nucleic acid that can be expressed by a recombinant transcription unit contained in a cell or in a cell-free transcription and translation system. Such sequences can be provided in the form of an open reading frame that is not interrupted by an internal non-translated sequence or intron, which is usually present in a eukaryotic gene. Genomic DNA containing related sequences can also be used to form a recombinant gene or transcription unit. The sequence of non-translated DNA can be present at the 5' end or 3' end of the open reading frame, wherein such sequences do not interfere with the manipulation or expression of the coding region, and can actually play a role in regulating the production of the desired product by various mechanisms (see "DNA regulatory sequence" below).

[0041] Therefore, for example, the term "recombinant" polynucleotide or "recombinant" nucleic acid refers to a non-naturally occurring polynucleotide or nucleic acid, such as a polynucleotide or nucleic acid made by an artificial combination of two otherwise separated segments of a sequence through human intervention. This artificial combination is often accomplished by chemical synthesis means or by artificially manipulating a separate segment of a nucleic acid (e.g., by genetic engineering techniques). This operation is usually performed to replace codons with redundant codons encoding the same or conservative amino acids, while sequence recognition sites are usually introduced or removed. Alternatively, this operation is performed to connect nucleic acid segments with desired functions together to produce a desired functional combination. This artificial combination is often accomplished by chemical synthesis means or by artificially manipulating a separate segment of a nucleic acid (e.g., by genetic engineering techniques).

[0042] Similarly, the term "recombinant" polypeptide refers to a non-naturally occurring polypeptide, such as a polypeptide made by the artificial combination of two otherwise separate segments of amino acid sequence through human intervention. Thus, for example, a polypeptide comprising a heterologous amino acid sequence is recombinant.

[0043] "Construct" or "vector" means a recombinant nucleic acid, generally recombinant DNA, which is generated for the purpose of expressing and / or propagating one or more specific nucleotide sequences, or for constructing other recombinant nucleotide sequences.

[0044] 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, which provide for and / or regulate the expression of a coding sequence and / or the production of the encoded polypeptide in a host cell.

[0045] The term "conversion" and "genetic modification" are used interchangeably herein, and refer to permanent or transient genetic changes induced in the cell after introducing new nucleic acids (i.e., DNA exogenous to the cell). Genetic changes ("modifications") can be completed by introducing new nucleic acids into the genome of the host cell or by transient or stable maintenance of new nucleic acids as episomal elements. When the cell is a eukaryotic cell, permanent genetic changes are generally completed by introducing new DNA into the genome of the cell. In prokaryotic cells, permanent changes can be introduced into chromosomes or introduced into chromosomes via extrachromosomal elements (such as plasmids and expression vectors), which may contain one or more selectable markers to help them maintain in recombinant host cells. Suitable methods for genetic modification include viral infection, transfection, conjugation, protoplast fusion, electroporation, particle gun technology, calcium phosphate precipitation, direct microinjection, etc. The selection of methods generally depends on the type of cells being transformed and the environment (i.e., in vitro, ex vivo, or in vivo) where the transformation occurs. A general discussion of these methods can be found in Ausubel et al., Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995.

[0046] "Operably linked" refers to a juxtaposition in which the components are in a relationship that permits them to 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. As used herein, the terms "heterologous promoter" and "heterologous control regions" refer to promoters and other control regions that are not normally associated with a particular nucleic acid in nature. For example, a "transcriptional control region heterologous to a coding region" is a transcriptional control region that is not normally associated with a coding region in nature.

[0047] As used herein, "host cell" refers to a cell (e.g., cell line) from a multicellular organism cultured as a unicellular entity, either in vivo or in vitro, a eukaryotic cell, a prokaryotic cell, or as a unicellular entity, which can be used as or has been used as a receptor for nucleic acids (e.g., expression vectors), and includes progeny of the original cell that has been genetically modified by nucleic acids. It should be understood that due to natural, accidental or intentional mutations, the progeny of the unicellular cell may not necessarily be identical to the original parent in morphology or in terms of genome or total DNA complementary sequence. A "recombinant host cell" (also referred to as a "genetically modified host cell") is a host cell into which a heterologous nucleic acid (e.g., expression vector) has been introduced. For example, a subject prokaryotic host cell is a prokaryotic host cell (e.g., bacteria) that has been genetically modified by introducing a heterologous nucleic acid, e.g., an exogenous nucleic acid that is foreign to the prokaryotic host cell (not normally found in nature) or a recombinant nucleic acid that is not normally found in a prokaryotic host cell, into a suitable prokaryotic host cell; and a subject eukaryotic host cell is a eukaryotic host cell that has been genetically modified by introducing a heterologous nucleic acid, e.g., an exogenous nucleic acid that is foreign to the eukaryotic host cell or a recombinant nucleic acid that is not normally found in a eukaryotic host cell, into a suitable eukaryotic host cell.

[0048] The term "conservative amino acid substitution" refers to the interchangeability of amino acid residues with similar side chains in proteins. For example, a group of amino acids with aliphatic side chains consists of glycine, alanine, valine, leucine, and isoleucine; a group of amino acids with aliphatic-hydroxy 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; 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, and asparagine-glutamine.

[0049] A polynucleotide or polypeptide has a certain percentage of "sequence identity" with another polynucleotide or polypeptide, which means that the percentage of bases or amino acids is the same when aligned, and the bases or amino acids are in the same relative position when the two sequences are compared. Sequence similarity can be determined in many different ways. To determine sequence identity, methods and computer programs including BLAST, which can be obtained through the World Wide Web ncbi.nlm.nih.gov / BLAST, can be used to align sequences. See, for example, Altschul et al. (1990), J. Mol. Biol. 215: 403-10. Another alignment algorithm is FASTA, which is available from the Genetic Computing Group (GCG) program package of a wholly owned subsidiary of Oxford Molecular Group, Inc. in Madison, Wisconsin, USA. Other techniques for alignment are described in Methods in Enzymology, Vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), Doolittle, ed., Academic Press, Inc., a division of Harcourt Brace & Co., San Diego, California, USA. Of particular interest are alignment programs that allow for gaps in the sequence. Smith-Waterman is a type of algorithm that allows for gaps in sequence alignments. See Meth. Mol. Biol. 70: 173-187 (1997). In addition, the GAP program using the Needleman and Wunsch alignment method can be used to align sequences. See J. Mol. Biol. 48: 443-453 (1970).

[0050] As used herein, the terms "treatment," "treating," and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect may be preventive, in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic, in terms of partially or completely curing a disease and / or side effects attributable to the disease. As used herein, "treatment" encompasses any treatment of a disease in a mammal (e.g., a human), and includes: (a) preventing the occurrence of a disease in a subject who may be susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, i.e., arresting its development; and (c) alleviating the disease, i.e., causing regression of the disease.

[0051] The terms "individual," "subject," "host," and "patient," used interchangeably herein, refer to an individual organism, such as a mammal, including but not limited to rodents, apes, non-human primates, humans, mammalian farm animals, mammalian sport animals, and mammalian pets.

[0052] Before further describing the present invention, it should be understood that the present invention is not limited to the specific embodiments described, and therefore, of course, may vary. It should also be understood that the terminology used herein is only for the purpose of describing specific embodiments, and is not intended to be limiting, because the scope of the present invention will only be limited by the appended claims.

[0053] Where a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of this range (unless the context clearly indicates otherwise, otherwise accurate to one tenth of the unit of the lower limit), as well as any other stated or intervening values ​​in this stated range, are encompassed within the present invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the present invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the stated limits, ranges excluding either or both of those included limits are also encompassed within the present invention.

[0054] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the invention 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 invention, preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe methods and / or materials in conjunction with the cited publications.

[0055] It must be noted that, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "CasZ polypeptide" includes a plurality of such polypeptides, and reference to a "guide RNA" includes reference to one or more guide RNAs and their equivalents known to those of skill in the art, etc. It should also be noted that the claims may be drafted to exclude any optional elements. Thus, this statement is intended to serve as a prerequisite basis for using exclusive terminology such as "solely", "only", etc. in conjunction with the recitation of claim elements, or for using a "negative" limitation.

[0056] It should be understood that certain features of the present invention described in the context of separate embodiments for clarity purposes may also be provided in combination in a single embodiment. Conversely, various features of the present invention described in the context of a single embodiment for simplicity may also be provided separately or in any suitable sub-combination. All combinations belonging to embodiments of the present invention are specifically encompassed in the present invention and disclosed herein as if each and every combination were individually and clearly disclosed. In addition, all sub-combinations of various embodiments and elements thereof are also specifically encompassed in the present invention and disclosed herein as if each and every such sub-combination were individually and clearly disclosed herein.

[0057] The publications discussed herein are provided only as of their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to precede such publications by virtue of prior invention. In addition, the publication date provided may be different from the actual publication date which may require independent confirmation. DETAILED DESCRIPTION

[0058] The present disclosure provides compositions and methods comprising one or more of the following: (1) a "CasZ" protein (also referred to as a CasZ polypeptide), a nucleic acid encoding a CasZ protein, and / or a modified host cell comprising a CasZ protein (and / or a nucleic acid encoding the CasZ protein); (2) a CasZ guide RNA that binds to a CasZ protein and provides sequence specificity for the CasZ protein, a nucleic acid encoding a CasZ guide RNA, and / or a modified host cell comprising a CasZ guide RNA (and / or a nucleic acid encoding the CasZ guide RNA); and (3) a CasZ trans-activating noncoding RNA (trancRNA) (referred to herein as "CasZtrancRNA"), a nucleic acid encoding a CasZtrancRNA, and / or a modified host cell comprising a CasZtrancRNA (and / or a nucleic acid encoding the CasZtrancRNA).

[0059] Composition

[0060] CRISPR / CASZ proteins, guide RNAs and transRNAs

[0061] 2 types of CRISPR-Cas systems are characterized by including an effector module of a single multi-domain protein. In the CasZ system, CRISPR / Cas endonucleases (e.g., CasZ proteins) interact (combine) with corresponding guide RNAs (e.g., CasZ guide RNAs) to form ribonucleoprotein (RNP) complexes, which are targeted to specific sites in target nucleic acids by base pairing between guide RNAs and target sequences in target nucleic acid molecules. Guide RNAs include nucleotide sequences (guide sequences) complementary to the sequences (target sites) of target nucleic acids. Therefore, CasZ proteins form complexes with CasZ guide RNAs, and guide RNAs provide sequence specificity for RNP complexes by guide sequences. The CasZ proteins of complexes provide site-specific activity. In other words, CasZ proteins are directed to target nucleic acids (e.g., target nucleotide sequences in target chromosome nucleic acids due to their association with guide RNAs; or target nucleotide sequences in nucleic acids outside target chromosomes, such as free nucleic acids, microcircle nucleic acids, mitochondrial nucleic acids, chloroplast nucleic acids, etc.) in target sites (e.g., stabilized in target sites).

[0062] The present disclosure provides compositions comprising CasZ polypeptides (and / or nucleic acids encoding CasZ polypeptides) (e.g., wherein the CasZ polypeptides can be naturally occurring CasZ proteins, nickase CasZ proteins, dCasZ proteins, chimeric CasZ proteins, etc.) (CasZa, CasZb, CasZc, CasZd, CasZe, CasZf, CasZg, CasZh, CasZi, CasZj, CasZK or CasZl proteins). The present disclosure provides compositions comprising CasZ guide RNAs (and / or nucleic acids encoding CasZ guide RNAs). For example, the present disclosure provides compositions comprising (a) CasZ polypeptides (and / or nucleic acids encoding CasZ polypeptides) and (b) CasZ guide RNAs (and / or nucleic acids encoding CasZ guide RNAs). The present disclosure provides a nucleic acid / protein complex (RNP complex), the nucleic acid / protein complex comprising: (a) CasZ polypeptides; and (b) CasZ guide RNAs. The present disclosure provides compositions comprising CasZ trancRNAs. The present disclosure provides a composition comprising CasZ trancRNA and one or more of the following: (a) CasZ protein and (b) CasZ guide RNA (e.g., comprising CasZ trancRNA and CasZ protein, CasZ trancRNA and CasZ guide RNA, or CasZ trancRNA and CasZ protein and CasZ guide RNA. The present disclosure provides a nucleic acid / protein complex (RNP complex), comprising: (a) CasZ polypeptide; (b) CasZ guide RNA; and (c) CasZ trancRNA. The present disclosure provides a composition comprising a CasZ protein and one or more of the following: (a) CasZ trancRNA and (b) CasZ guide RNA.

[0063] Cas9 protein

[0064] CasZ polypeptides (this term is used interchangeably with the terms "CasZ protein", "Cas14", "Cas14 polypeptide" or "Cas14 protein") can bind and / or modify (e.g., cut, 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 CasZ protein includes an active fusion partner, and in some cases, the CasZ protein provides nuclease activity). In some cases, the CasZ protein is a naturally occurring protein (e.g., naturally occurring in prokaryotic cells). In other cases, the CasZ protein is not a naturally occurring polypeptide (e.g., the CasZ protein is a variant CasZ protein, a chimeric protein, etc.). The CasZ protein includes three partial RuvC domains (RuvC-I, RuvC-II and RuvC-III, also referred to herein as subdomains), which are discontinuous relative to the primary amino acid sequence of the CasZ protein, but once the protein is produced and folded, the RuvC domain is formed. The naturally occurring CasZ protein acts as an endonuclease, catalyzing cleavage at a specific sequence in a targeted nucleic acid (e.g., double-stranded dsDNA). Sequence specificity is provided by an associated guide RNA, which hybridizes with a target sequence within the target DNA. The naturally occurring CasZ guide RNA is a crRNA, wherein the crRNA includes (i) a guide sequence that hybridizes with a target sequence in the target DNA and (ii) a protein binding segment that binds to the CasZ protein.

[0065] In some embodiments, the CasZ protein of the subject methods and / or compositions is (or is derived from) a naturally occurring (wild-type) protein. Examples of naturally occurring CasZ proteins (e.g., CasZa, CasZb, CasZc, CasZd, CasZe, CasZf, CasZg, CasZh, CasZi, CasZj, CasZk, CasZl) are described in Figure 1In some cases, the subject CasZ protein is a CasZa protein. In some cases, the subject CasZ protein is a CasZb protein. In some cases, the subject CasZ protein is a CasZc protein. In some cases, the subject CasZ protein is a CasZd protein. In some cases, the subject CasZ protein is a CasZe protein. In some cases, the subject CasZ protein is a CasZf protein. In some cases, the subject CasZ protein is a CasZg protein. In some cases, the subject CasZ protein is a CasZh protein. In some cases, the subject CasZ protein is a CasZi protein. In some cases, the subject CasZ protein is a CasZj protein. In some cases, the subject CasZ protein is a CasZk protein. In some cases, the subject CasZ protein is a CasZl protein. In some cases, the subject CasZ protein is a CasZe, CasZf, CasZg, or CasZh protein. In some cases, the subject CasZ protein is a CasZj, CasZk, or CasZl protein.

[0066] It is important to note that this newly discovered protein (CasZ) is shorter than previously identified CRISPR-Cas endonucleases, and therefore using this protein as an alternative provides the advantage that the nucleotide sequence encoding the protein is relatively short. For example, in cases where a nucleic acid encoding a CasZ protein is required, such as in the case of a viral vector (e.g., AAV vector), this can be used for delivery to cells such as eukaryotic cells (e.g., mammalian cells, human cells, mouse cells, in vitro, ex vivo, in vivo) for research and / or clinical applications. In addition, in its natural environment, the DNA sequence encoding CasZ is present in a locus that also has a Cas1 protein.

[0067] In some cases, the subject CasZ protein has a length of 900 amino acids or less (e.g., 850 amino acids or less, 800 amino acids or less, 750 amino acids or less, or 700 amino acids or less). In some cases, the subject CasZ protein has a length of 850 amino acids or less (e.g., 850 amino acids or less). In some cases, the subject CasZ protein has a length of 800 amino acids or less (e.g., 750 amino acids or less). In some cases, the subject CasZ protein has a length of 700 amino acids or less. In some cases, the subject CasZ protein has a length of 650 amino acids or less.

[0068] In some cases, a subject CasZ protein has a length in the range of 350-900 amino acids (e.g., 350-850, 350-800, 350-750, 350-700, 400-900, 400-850, 400-800, 400-750, or 400-700 amino acids).

[0069] In some cases, the subject CasZ protein (e.g., CasZa) has a length in the range of 350-750 amino acids (e.g., 350-700, 350-550, 450-550, 450-750, 450-650, or 450-550 amino acids). In some cases, the subject CasZ protein (e.g., CasZa) has a length in the range of 450-750 amino acids (e.g., 500-700 amino acids). In some cases, the subject CasZ protein (e.g., CasZa) has a length in the range of 350-700 amino acids (e.g., 350-650, 350-600, or 350-550 amino acids). In some cases, the subject CasZ protein (e.g., CasZa) has a length in the range of 500-700 amino acids. In some cases, the subject CasZ protein (e.g., CasZa) has a length in the range of 450-550 amino acids. In some cases, the subject CasZ protein (e.g., CasZa) has a length in the range of 350-550 amino acids.

[0070] In some cases, the subject CasZ protein (e.g., CasZb) has a length in the range of 350-700 amino acids (e.g., 350-650 or 350-620 amino acids). In some cases, the subject CasZ protein (e.g., CasZb) has a length in the range of 450-700 amino acids (e.g., 450-650, 500-650 or 500-620 amino acids). In some cases, the subject CasZ protein (e.g., CasZb) has a length in the range of 500-650 amino acids (e.g., 500-620 amino acids). In some cases, the subject CasZ protein (e.g., CasZb) has a length in the range of 500-620 amino acids.

[0071] In some cases, the subject CasZ protein (e.g., CasZc) has a length in the range of 600-800 amino acids (e.g., 600-650 or 700-800 amino acids). In some cases, the subject CasZ protein (e.g., CasZc) has a length in the range of 600-650 amino acids. In some cases, the subject CasZ protein (e.g., CasZc) has a length in the range of 700-800 amino acids.

[0072] In some cases, the subject CasZ protein (e.g., CasZd) has a length in the range of 400-650 amino acids (e.g., 400-600, 400-550, 500-650, 500-600, or 500-550 amino acids). In some cases, the subject CasZ protein (e.g., CasZd) has a length in the range of 500-600 amino acids. In some cases, the subject CasZ protein (e.g., CasZd) has a length in the range of 500-550 amino acids. In some cases, the subject CasZ protein (e.g., CasZd) has a length in the range of 400-550 amino acids.

[0073] In some cases, the subject CasZ protein (e.g., CasZe) has a length in the range of 450-700 amino acids (e.g., 450--650, 450-615, 475-700, 475-650, or 475-615 amino acids). In some cases, the subject CasZ protein (e.g., CasZe) has a length in the range of 450-675 amino acids. In some cases, the subject CasZ protein (e.g., CasZe) has a length in the range of 475-675 amino acids.

[0074] In some cases, the subject CasZ protein (e.g., CasZf) has a length in the range of 400-550 amino acids (e.g., 400-520, 400-500, 400-475, 415-550, 415-520, 415-500, or 415-475 amino acids). In some cases, the subject CasZ protein (e.g., CasZf) has a length in the range of 400-475 amino acids (e.g., 400-450 amino acids).

[0075] In some cases, the subject CasZ protein (e.g., CasZg) has a length in the range of 500-750 amino acids (e.g., 550-750 or 500-700 amino acids). In some cases, the subject CasZ protein (e.g., CasZg) has a length in the range of 700-750 amino acids. In some cases, the subject CasZ protein (e.g., CasZg) has a length in the range of 550-600 amino acids.

[0076] In some cases, the subject CasZ protein (e.g., CasZh) has a length in the range of 380-450 amino acids (e.g., 380-420, 400-450, or 400-420 amino acids). In some cases, the subject CasZ protein (e.g., CasZh) has a length in the range of 400-420 amino acids.

[0077] In some cases, the subject CasZ protein (e.g., CasZi) has a length in the range of 700-800 amino acids (e.g., 700-750, 720-800, or 720-750 amino acids). In some cases, the subject CasZ protein (e.g., CasZi) has a length in the range of 720-780 amino acids.

[0078] In some cases, the subject CasZ protein (e.g., CasZj) has a length in the range of 600-750 amino acids (e.g., 600-700 or 650-700 amino acids). In some cases, the subject CasZ protein (e.g., CasZj) has a length in the range of 400-420 amino acids.

[0079] In some cases, the subject CasZ protein (e.g., CasZk) has a length in the range of 450-600 amino acids (e.g., 450-580, 480-600, 480-580, or 500-600 amino acids). In some cases, the subject CasZ protein (e.g., CasZk) has a length in the range of 480-580 amino acids.

[0080] In some cases, the subject CasZ protein (e.g., CasZ1) has a length in the range of 350-500 amino acids (e.g., 350-450, 380-450, 350-420, or 380-420 amino acids). In some cases, the subject CasZ protein (e.g., CasZ1) has a length in the range of 380-420 amino acids.

[0081] In some cases, a subject CasZ protein (of a subject composition and / or method) comprises Figure 1 or Figure 7 The subject CasZa protein has an amino acid sequence with 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). For example, in some cases, the subject CasZa protein comprises an amino acid sequence with Figure 1 or Figure 7 The subject CasZa protein has an amino acid sequence with 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). In some cases, the subject CasZa protein comprises an amino acid sequence with Figure 1 or Figure 7 The subject CasZa protein has an amino acid sequence with 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). In some cases, the subject CasZa protein comprises an amino acid sequence with Figure 1 or Figure 7 The CasZa protein has an amino acid sequence with 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity). In some cases, the subject CasZa protein comprises Figure 1 or Figure 7 In some cases, the subject CasZ protein comprises Figure 1 or Figure 7 The subject CasZa amino acid sequence is different from that of the subject CasZa amino acid sequence, except that the sequence comprises one or more amino acid substitutions (e.g., 1, 2, or 3 amino acid substitutions) that reduce the naturally occurring catalytic activity of the protein (e.g., such as, for example, at one or more catalytic amino acid positions) (e.g., in some cases, such that the CasZ protein is dCasZ). In some cases, the subject CasZ protein comprises Figure 1 or Figure 7The CasZa protein has an amino acid sequence with 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) and has a length in the range of 350-800 amino acids (e.g., 350-800, 350-750, 350-700, 350-550, 450-550, 450-750, 450-650, or 450-550 amino acids).

[0082] In some cases, a subject CasZ protein (of a subject composition and / or method) comprises Figure 1 or Figure 7 The CasZb protein of the subject invention has an amino acid sequence with 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). For example, in some cases, the subject CasZ protein comprises an amino acid sequence with Figure 1 or Figure 7 The subject CasZb protein has an amino acid sequence with 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). In some cases, the subject CasZ protein comprises an amino acid sequence with Figure 1 or Figure 7 The CasZb protein of the subject invention has an amino acid sequence with 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). In some cases, the subject CasZ protein comprises an amino acid sequence with Figure 1 or Figure 7 The CasZb protein of the subject CasZb has an amino acid sequence with 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity). In some cases, the subject CasZ protein comprises Figure 1 or Figure 7 In some cases, the subject CasZ protein comprises Figure 1 or Figure 7In some cases, the subject CasZ protein comprises a CasZb amino acid sequence, except that the sequence comprises an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein (e.g., such as, for example, at one or more catalytic amino acid positions) (e.g., in some cases, such that the CasZ protein is dCasZ). Figure 1 or Figure 7 The CasZb protein has an amino acid sequence with 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) and has a length in the range of 350-700 amino acids (e.g., 350-650 or 350-620 amino acids).

[0083] In some cases, a subject CasZ protein (of a subject composition and / or method) comprises Figure 1 or Figure 7 The CasZc protein of the subject invention has an amino acid sequence with 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). For example, in some cases, the subject CasZ protein comprises an amino acid sequence with Figure 1 or Figure 7 The subject CasZc protein has an amino acid sequence with 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). In some cases, the subject CasZ protein comprises an amino acid sequence with Figure 1 or Figure 7 The CasZc protein of the subject 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). In some cases, the subject CasZ 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). Figure 1 or Figure 7 The CasZc protein of the subject has an amino acid sequence with 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity). In some cases, the subject CasZ protein comprises Figure 1 or Figure 7 In some cases, the subject CasZ protein comprises Figure 1 or Figure 7A CasZc amino acid sequence of the invention, except that the sequence comprises an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein (e.g., such as, for example, at one or more catalytic amino acid positions) (e.g., in some cases, such that the CasZ protein is dCasZ). In some cases, the subject CasZ protein comprises Figure 1 or Figure 7 The CasZc protein has an amino acid sequence with 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) and has a length in the range of 600-800 amino acids (e.g., 600-650 or 700-800 amino acids).

[0084] In some cases, a subject CasZ protein (of a subject composition and / or method) comprises Figure 1 or Figure 7 The CasZd protein of the subject invention has an amino acid sequence with 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). For example, in some cases, the subject CasZ protein comprises an amino acid sequence with Figure 1 or Figure 7 The subject CasZd protein has an amino acid sequence with 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). In some cases, the subject CasZ protein comprises an amino acid sequence with Figure 1 or Figure 7 The CasZd protein of the subject 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). In some cases, the subject CasZ 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). Figure 1 or Figure 7 The CasZd protein of the subject has an amino acid sequence with 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity). In some cases, the subject CasZ protein comprises Figure 1 or Figure 7 In some cases, the subject CasZ protein comprises Figure 1 or Figure 7In some cases, the subject CasZ protein comprises a CasZd amino acid sequence, except that the sequence comprises an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein (e.g., such as, for example, at one or more catalytic amino acid positions) (e.g., in some cases, such that the CasZ protein is a dCasZ). Figure 1 or Figure 7 The CasZd protein has an amino acid sequence with 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) and has a length in the range of 400-650 amino acids (e.g., 400-600, 400-550, 500-650, 500-600, or 500-550 amino acids).

[0085] In some cases, a subject CasZ protein (of a subject composition and / or method) comprises Figure 1 or Figure 7 The CasZe protein of the present invention has an amino acid sequence with 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). For example, in some cases, the subject CasZ protein comprises an amino acid sequence with Figure 1 or Figure 7 The subject CasZe protein has an amino acid sequence with 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). In some cases, the subject CasZe protein comprises an amino acid sequence with Figure 1 or Figure 7 The CasZe protein has an amino acid sequence with 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). In some cases, the subject CasZ protein comprises an amino acid sequence with Figure 1 or Figure 7 The CasZe protein has an amino acid sequence with 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity). In some cases, the subject CasZe protein comprises Figure 1 or Figure 7 In some cases, the subject CasZ protein comprises Figure 1 or Figure 7 A CasZe amino acid sequence of the invention, except that the sequence comprises an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein (e.g., such as, for example, at one or more catalytic amino acid positions) (e.g., in some cases, such that the CasZ protein is dCasZ). In some cases, the subject CasZ protein comprises Figure 1 or Figure 7 The Cas9 protein has an amino acid sequence with 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) and has a length ranging from 450-700 amino acids (e.g., 450-650, 450-615, 475-700, 475-650, or 475-615 amino acids).

[0086] In some cases, a subject CasZ protein (of a subject composition and / or method) comprises Figure 1 or Figure 7 The CasZf protein of the present invention has an amino acid sequence with 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). For example, in some cases, the subject CasZ protein comprises an amino acid sequence with Figure 1 or Figure 7 The subject CasZ 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) with the subject CasZ protein. Figure 1 or Figure 7 The CasZf protein of the present invention has an amino acid sequence with 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). In some cases, the subject CasZ protein comprises an amino acid sequence with Figure 1 or Figure 7 The CasZf protein has an amino acid sequence with 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity). In some cases, the subject CasZ protein comprises Figure 1 or Figure 7In some cases, the subject CasZ protein comprises Figure 1 or Figure 7 A CasZf amino acid sequence of the subject CasZ protein, except that the sequence comprises an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein (e.g., such as, for example, at one or more catalytic amino acid positions) (e.g., in some cases, such that the CasZ protein is dCasZ). In some cases, the subject CasZ protein comprises Figure 1 or Figure 7 The CasZf protein has an amino acid sequence with 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) and has a length in the range of 400-750 amino acids (e.g., 400-700, 700-650, 400-620, 400-600, 400-550, 400-520, 400-500, 400-475, 415-550, 415-520, 415-500, or 415-475 amino acids).

[0087] In some cases, a subject CasZ protein (of a subject composition and / or method) comprises Figure 1 or Figure 7 The CasZg protein of the subject invention has an amino acid sequence with 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). For example, in some cases, the subject CasZ protein comprises an amino acid sequence with Figure 1 or Figure 7 The subject CasZg protein has an amino acid sequence with 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). In some cases, the subject CasZ protein comprises an amino acid sequence with Figure 1 or Figure 7 The CasZg protein of the subject 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). In some cases, the subject CasZ 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). Figure 1 or Figure 7The CasZg protein has an amino acid sequence with 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity). In some cases, the subject CasZ protein comprises Figure 1 or Figure 7 In some cases, the subject CasZ protein comprises Figure 1 or Figure 7 A CasZg amino acid sequence of a subject CasZ protein, except that the sequence comprises an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein (e.g., such as, for example, at one or more catalytic amino acid positions) (e.g., in some cases, such that the CasZ protein is dCasZ). In some cases, the subject CasZ protein comprises Figure 1 or Figure 7 The CasZg protein has an amino acid sequence with 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) and has a length in the range of 500-750 amino acids (e.g., 500-750 amino acids (e.g., 550-750 amino acids)).

[0088] In some cases, a subject CasZ protein (of a subject composition and / or method) comprises Figure 1 or Figure 7 The CasZh protein of the subject invention has an amino acid sequence with 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). For example, in some cases, the subject CasZ protein comprises an amino acid sequence with Figure 1 or Figure 7 The subject CasZh protein has an amino acid sequence with 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). In some cases, the subject CasZ protein comprises an amino acid sequence with Figure 1 or Figure 7 The CasZh protein of the subject invention has an amino acid sequence with 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). In some cases, the subject CasZ protein comprises an amino acid sequence with Figure 1 or Figure 7The CasZh protein has an amino acid sequence with 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity). In some cases, the subject CasZ protein comprises Figure 1 or Figure 7 In some cases, the subject CasZ protein comprises Figure 1 or Figure 7 A CasZh amino acid sequence of a subject CasZh, except that the sequence comprises an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein (e.g., such as, for example, at one or more catalytic amino acid positions) (e.g., in some cases, such that the CasZ protein is dCasZ). In some cases, the subject CasZ protein comprises Figure 1 or Figure 7 The CasZh protein has an amino acid sequence with 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) and has a length in the range of 380-450 amino acids (e.g., 380-420, 400-450, or 400-420 amino acids).

[0089] In some cases, a subject CasZ protein (of a subject composition and / or method) comprises Figure 1 or Figure 7 The CasZi protein of the subject may have an amino acid sequence with 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). For example, in some cases, the subject CasZ protein comprises an amino acid sequence with Figure 1 or Figure 7 The subject CasZi protein has an amino acid sequence with 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). In some cases, the subject CasZ protein comprises an amino acid sequence with Figure 1 or Figure 7 The CasZi protein of the subject has an amino acid sequence with 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). In some cases, the subject CasZ protein comprises an amino acid sequence with Figure 1 or Figure 7The CasZi protein has an amino acid sequence with 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity). In some cases, the subject CasZ protein comprises Figure 1 or Figure 7 In some cases, the subject CasZ protein comprises Figure 1 or Figure 7 A CasZi amino acid sequence of the subject invention, except that the sequence comprises an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein (e.g., such as, for example, at one or more catalytic amino acid positions) (e.g., in some cases, such that the CasZ protein is dCasZ). In some cases, the subject CasZ protein comprises Figure 1 or Figure 7 The CasZi protein has an amino acid sequence with 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) and has a length in the range of 700-800 amino acids (e.g., 700-750, 720-800, or 720-750 amino acids).

[0090] In some cases, a subject CasZ protein (of a subject composition and / or method) comprises Figure 1 or Figure 7 The CasZj protein of the subject invention has an amino acid sequence with 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). For example, in some cases, the subject CasZ protein comprises an amino acid sequence with Figure 1 or Figure 7 The subject CasZj protein has an amino acid sequence with 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). In some cases, the subject CasZ protein comprises an amino acid sequence with Figure 1 or Figure 7 The CasZj protein has an amino acid sequence with 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). In some cases, the subject CasZ protein comprises an amino acid sequence with Figure 1 or Figure 7The CasZj protein has an amino acid sequence with 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity). In some cases, the subject CasZ protein comprises Figure 1 or Figure 7 In some cases, the subject CasZ protein comprises Figure 1 or Figure 7 A CasZj amino acid sequence of the subject CasZ protein, except that the sequence comprises an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein (e.g., such as, for example, at one or more catalytic amino acid positions) (e.g., in some cases, such that the CasZ protein is dCasZ). In some cases, the subject CasZ protein comprises Figure 1 or Figure 7 The CasZj protein has an amino acid sequence with 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) and has a length in the range of 600-750 amino acids (e.g., 600-700 or 650-700 amino acids).

[0091] In some cases, a subject CasZ protein (of a subject composition and / or method) comprises Figure 1 or Figure 7 The CasZk protein of the present invention has an amino acid sequence with 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). For example, in some cases, the subject CasZ protein comprises an amino acid sequence with Figure 1 or Figure 7 The subject CasZk protein has an amino acid sequence with 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). In some cases, the subject CasZ protein comprises an amino acid sequence with Figure 1 or Figure 7 The subject CasZk protein has an amino acid sequence with 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). In some cases, the subject CasZ protein comprises an amino acid sequence with Figure 1 or Figure 7The CasZk protein of the subject has an amino acid sequence with 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity). In some cases, the subject CasZ protein comprises Figure 1 or Figure 7 In some cases, the subject CasZ protein comprises Figure 1 or Figure 7 A CasZk amino acid sequence of a subject CasZk protein, except that the sequence comprises an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein (e.g., such as, for example, at one or more catalytic amino acid positions) (e.g., in some cases, such that the CasZ protein is dCasZ). In some cases, the subject CasZ protein comprises Figure 1 or Figure 7 The CasZk protein has an amino acid sequence with 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) and has a length in the range of 450-600 amino acids (e.g., 450-580, 480-600, 480-580, or 500-600 amino acids).

[0092] In some cases, a subject CasZ protein (of a subject composition and / or method) comprises Figure 1 or Figure 7 The subject CasZ1 protein has an amino acid sequence with 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). For example, in some cases, the subject CasZ protein comprises an amino acid sequence with Figure 1 or Figure 7 The subject CasZ1 protein has an amino acid sequence with 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). In some cases, the subject CasZ protein comprises an amino acid sequence with Figure 1 or Figure 7 The subject CasZ1 protein has an amino acid sequence with 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). In some cases, the subject CasZ protein comprises an amino acid sequence with Figure 1 or Figure 7 The CasZ1 protein of the subject has an amino acid sequence with 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity). In some cases, the subject CasZ protein comprises Figure 1 or Figure 7 In some cases, the subject CasZ protein comprises Figure 1 or Figure 7 In some cases, the subject CasZ protein comprises a CasZ1 amino acid sequence, except that the sequence comprises an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein (e.g., such as, for example, at one or more catalytic amino acid positions) (e.g., in some cases, such that the CasZ protein is dCasZ). Figure 1 or Figure 7 The CasZ1 protein has an amino acid sequence with 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) and has a length in the range of 450-600 amino acids (e.g., 450-580, 480-600, 480-580, or 500-600 amino acids).

[0093] In some cases, a subject CasZ protein (of a subject composition and / or method) comprises Figure 1 or Figure 7 The subject CasZe, CasZf, CasZg, or CasZh protein has an amino acid sequence with 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). For example, in some cases, the subject CasZ protein comprises an amino acid sequence with Figure 1 or Figure 7 CasZe, CasZf, CasZg, or CasZh protein 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) amino acid sequence. In some cases, the subject CasZ 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). Figure 1 or Figure 7The subject CasZe, CasZf, CasZg, or CasZh protein has an amino acid sequence with 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). In some cases, the subject CasZ protein comprises an amino acid sequence with Figure 1 or Figure 7 The subject CasZe, CasZf, CasZg, or CasZh protein has an amino acid sequence with 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity). In some cases, the subject CasZ protein comprises an amino acid sequence with Figure 1 or Figure 7 In some cases, the subject CasZ protein comprises an amino acid sequence having Figure 1 or Figure 7 The amino acid sequence of a CasZe, CasZf, CasZg or CasZh protein sequence of the subject CasZe, CasZf, CasZg or CasZh protein sequence, except that the sequence comprises an amino acid substitution (e.g., 1, 2 or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein (e.g., such as, for example, at one or more catalytic amino acid positions). In some cases, the subject CasZ protein comprises Figure 1 or Figure 7 The CasZe, CasZf, CasZg or CasZh protein has an amino acid sequence with 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) and has a length in the range of 350-900 amino acids (e.g., 350-850, 350-800, 400-900, 400-850, or 400-800 amino acids).

[0094] In some cases, a subject CasZ protein (of a subject composition and / or method) comprises Figure 1 or Figure 7 The subject CasZa, CasZb, CasZc, CasZd, CasZe, CasZf, CasZg, CasZh, CasZi, CasZj, CasZK, or CasZl proteins have an amino acid sequence with 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). For example, in some cases, the subject CasZ protein comprises an amino acid sequence with Figure 1 or Figure 7 In some cases, the subject CasZ 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 a CasZa, CasZb, CasZc, CasZd, CasZe, CasZf, CasZg, CasZh, CasZi, CasZj, CasZK, or CasZl protein. Figure 1 or Figure 7 In some cases, the subject CasZ 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) with a CasZa, CasZb, CasZc, CasZd, CasZe, CasZf, CasZg, CasZh, CasZi, CasZj, CasZK, or CasZl protein. Figure 1 or Figure 7 In some cases, the subject CasZ 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 a CasZa, CasZb, CasZc, CasZd, CasZe, CasZf, CasZg, CasZh, CasZi, CasZj, CasZK, or CasZl protein. Figure 1 or Figure 7 In some cases, the subject CasZ protein comprises an amino acid sequence having Figure 1 or Figure 7 The amino acid sequence of a CasZa, CasZb, CasZc, CasZd, CasZe, CasZf, CasZg, CasZh, CasZi, CasZj, CasZK or CasZl protein sequence of the present invention, except that the sequence comprises an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein (such as, for example, at one or more catalytic amino acid positions). In some cases, the subject CasZ protein comprises Figure 1 or Figure 7The CasZa, CasZb, CasZc, CasZd, CasZe, CasZf, CasZg, CasZh, CasZi, CasZj, CasZK or CasZl protein has an amino acid sequence with 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) and has a length ranging from 350-900 amino acids (e.g., 350-850, 350-800, 400-900, 400-850, or 400-800 amino acids).

[0095] CasZ variants

[0096] When compared to the amino acid sequence of the corresponding wild-type CasZ protein, the variant CasZ protein has an amino acid sequence that differs in at least one amino acid (e.g., having a deletion, insertion, substitution, fusion). A CasZ protein that cuts one strand of a double-stranded target nucleic acid but does not cut the other strand is referred to herein as a "nickase" (e.g., "nickase CasZ"). A CasZ protein that is substantially free of nuclease activity is referred to herein as a dead CasZ protein ("dCasZ") (it should be noted that the nuclease activity can be provided by a heterologous polypeptide (fusion partner) in the case of a chimeric CasZ protein, which is described in more detail below). For any CasZ variant protein described herein (e.g., nickase CasZ, dCasZ, chimeric CasZ), the CasZ variant may include a CasZ protein sequence having the same parameters as described above (e.g., domains present, percent identity, etc.).

[0097] Variant – catalytic activity

[0098] In some cases, the CasZ protein is a variant CasZ protein, such as a protein that is mutated relative to a naturally occurring catalytically active sequence, and exhibits reduced cleavage activity (e.g., exhibiting 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, or 30% or less cleavage activity) when compared to the corresponding naturally occurring sequence. In some cases, such variant CasZ proteins are catalytically 'dead' proteins (substantially no cleavage activity) and may be referred to as 'dCasZ'. In some cases, the variant CasZ protein is a nickase (cutting only one strand of a double-stranded target nucleic acid (e.g., double-stranded target DNA)). As described in more detail herein, in some cases, a CasZ protein (in some cases, a CasZ protein having wild-type cleavage activity and in some cases, a variant CasZ having reduced cleavage activity, such as dCasZ or a nickase CasZ) is fused (conjugated) to a heterologous polypeptide having a target activity (e.g., a target catalytic activity) to form a fusion protein (chimeric CasZ protein).

[0099] When numbered according to CasZi.1, the catalytic residues of CasZ include D405, E586, and D684 (see, e.g., Figure 1 ). Thus, in some cases, the CasZ protein has reduced activity, and one or more of the above amino acids (or one or more corresponding amino acids of any CasZ protein) are mutated (e.g., substituted with alanine). In some cases, the variant CasZ protein is a catalytic 'dead' protein (catalytically inactive) and is referred to as 'dCasZ'. The dCasZ protein can be fused to a fusion partner that provides activity, and in some cases, dCasZ (e.g., a fusion partner that does not provide catalytic activity, but dCasZ that may have an NLS when expressed in a eukaryotic cell) can bind to the target DNA and can be used for imaging (e.g., the protein can be tagged / labeled), and / or RNA polymerase can be prevented from being transcribed from the target DNA. In some cases, the variant CasZ protein is a nickase (cuts only one strand of a double-stranded target nucleic acid (e.g., double-stranded target DNA)).

[0100] Variants – Chimeric CasZ (ie, fusion proteins)

[0101] As noted above, in some cases, a CasZ protein (in some cases, a CasZ protein having wild-type cleavage activity and in some cases, a variant CasZ having reduced cleavage activity, such as dCasZ or nickase CasZ) is fused (conjugated) to a heterologous polypeptide having a target activity (e.g., a target catalytic activity) to form a fusion protein (chimeric CasZ protein). The heterologous polypeptide to which the CasZ protein can be fused is referred to herein as a 'fusion partner'.

[0102] In some cases, the fusion partner can regulate the transcription of the target DNA (e.g., inhibit transcription, increase transcription). For example, in some cases, the fusion partner is a protein (or a domain from a protein) that inhibits transcription (e.g., a transcription repressor, a protein that works by the recruitment of transcription repressor proteins, modification of target DNA such as methylation, recruitment of DNA modifiers, regulation of histones associated with target DNA, recruitment of histone modifiers (such as those histone modifiers that modify the acetylation and / or methylation of histones)). In some cases, the fusion partner is a protein (or a domain from a protein) that increases transcription (e.g., a transcription activator, a protein that works by the recruitment of transcription activator proteins, modification of target DNA such as methylation, recruitment of DNA modifiers, regulation of histones associated with target DNA, recruitment of histone modifiers (such as those histone modifiers that modify the acetylation and / or methylation of histones)).

[0103] In some cases, the chimeric CasZ protein includes a heterologous polypeptide having an enzymatic activity that modifies a target nucleic acid (e.g., nuclease activity (such as FokI nuclease activity), methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination 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).

[0104] In some cases, the chimeric CasZ protein includes a heterologous polypeptide having an enzymatic activity (e.g., methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity, or demyristoylation activity) that modifies a polypeptide (e.g., a histone) associated with a target nucleic acid.

[0105] 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 subdomains (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 to 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.

[0106] 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 interacting domain (SID); ERF repressor domain (ERD), SRDX repressor domain (e.g., for repression in plants), etc.; histone lysine methyltransferases, such as Pr-SET7 / 8, SUV4-20H1, RIZ1, etc.; histone lysine demethylases, such as JMJD2A / JHDM3A, JMJD2B, JMJD2C / GASC1, JMJD2D, JARID1A / RBP2, JARID1B / PLU-1, JARID1C / SMCX, JARID1D / SMCY, etc.; histone lysine deacetylases, such as HDAC1, HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRT1, SIRT2, HDAC11, etc.; DNA methylases, such as HhaIDNA m5c-methyltransferase (M.HhaI), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3 (plants), ZMET2, CMT1, CMT2 (plants), etc.; and peripheral recruitment elements, such as lamin A, lamin B, etc.

[0107] In some cases, the fusion partner has an enzyme activity that modifies the target nucleic acid (e.g., ssRNA, dsRNA, ssDNA, dsDNA). Examples of enzyme activities that can be provided by the fusion partner include, but are not limited to: nuclease activity, such as activity provided by a restriction enzyme (e.g., FokI nuclease); methyltransferase activity, such as activity provided by a methyltransferase (e.g., HhaIDNAm5c-methyltransferase (M.HhaI), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3 (plant), ZMET2, CMT1, CMT2 (plant) and the like); demethylase activity, such as activity provided by a demethylase (e.g., 10-11 translocation (TET) dioxygenase 1 (TET1CD), TET1, DME, DML1, DML2, ROS1 and the like); DNA repair activity; DNA damage activity; deamination activity, such as the activity provided by a deaminase (e.g., a cytosine deaminase, such as rat APOBEC1); dismutase activity; alkylation activity; depurination activity; oxidation activity; pyrimidine dimer forming activity; integrase activity, such as the activity provided by an integrase and / or a resolvase (e.g., a Gin convertase such as an overactive mutant of Gin convertase GinH106Y, human immunodeficiency virus type 1 integrase (IN), Tn3 resolvase, etc.); transposase activity; recombinase activity, such as the activity provided by a recombinase (e.g., the catalytic domain of Gin recombinase); polymerase activity; ligase activity; helicase activity; photolyase activity and glycosylase activity).

[0108] In some cases, the fusion partner has an enzymatic activity that modifies a protein (eg, a histone, an RNA binding protein, a DNA binding protein, etc.) associated with a target nucleic acid (eg, ssRNA, dsRNA, ssDNA, dsDNA). Examples of enzymatic activities that can be provided by the fusion partner (modifying a protein associated with a target nucleic acid) include, but are not limited to: methyltransferase activity, such as that provided by a histone methyltransferase (HMT) (e.g., suppressor of mottled 3-9 homolog 1 (SUV39H1, also known as KMT1A), autosomal histone lysine methyltransferase 2 (G9A, also known as KMT1C and EHMT2), SUV39H2, ESET / SETDB1, etc., SET1A, SET1B, MLL1 to 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., the 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, activity provided by the enzymes 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; deribosylation activity; myristoylation activity and demyristoylation activity.

[0109] Additional examples of suitable fusion partners are dihydrofolate reductase (DHFR) destabilization domains (e.g., to produce chemically controllable chimeric CasZ proteins) and chloroplast transit peptides. Suitable chloroplast transit peptides include, but are not limited to:

[0110] MASMISSSAVTTVSRASRGQSAAMAPFGGLKSMTGFPVRKV NTDITSITSNGGRVKCMQVWPPIGKKKFETLSYLPPLTRDSRA

[0111] (SEQIDNO:101);

[0112] MASMISSSAVTTVSRASRGQSAAMAPFGGLKSMTGFPVRKV NTDITSITSNGGRVKS(SEQIDNO:102);

[0113] MASSMLSSATMVASPAQATMVAPFNGLKSSAAFPATRKANN DITSITSNGGRVNCMQVWPPIEKKKFETLSYLPDLTDSGGRVNC (SEQIDNO:103);

[0114] MAQVSRICNGVQNPSLISNLSKSSQRKSPLSVSLKTQQHPRAY PISSSWGLKKSGMTLIGSELRPLKVMSSVSTAC(SEQIDNO:104);

[0115] MAQVSRICNGVWNPSLISNLSKSSQRKSPLSVSLKTQQHPRA YPISSSWGLKKSGMTLIGSELRPLKVMSSVSTAC(SEQIDNO:105);

[0116] MAQINNMAQGIQTLNPNSNFHKPQVPKSSSFLVFGSKKLKNSANSMLVLKKDSIFMQLFCSFRISASVATAC(SEQIDNO:106);

[0117] MAALVTSQLATSGTVLSVTDRFRRPGFQGLRPRNPADAALGMRTVGASAAPKQSRKPHRFDRRCLSMVV(SEQIDNO:107);

[0118] MAALTTSQLATSATGFGIADRSAPSSLLRHGFQGLKPRSPAG GDATSLSVTTSARATPKQQRSVQRGSRRFPSVVVC(SEQIDNO: 108);

[0119] MASSVLSSAAVATRSNVAQANMVAPFTGLKSAASFPVSRKQ NLDITSIASNGGRVQC(SEQIDNO:109);

[0120] MESLAATSVFAPSRVAVPAARALVRAGTVVPTRRTSSTSGTS GVKCSAAVTPQASPVISRSAAAA (SEQ ID NO: 110); and

[0121] MGAAATSMQSLKFSNRLVPPSRRLSPVPNNVTCNNLPKSAAPVRTVKCCASSWNSTINGAAATTNGASAASS (SEQ ID NO: 111).

[0122] In some cases, the CasZ fusion polypeptide of the present disclosure comprises: a) a CasZ polypeptide of the present disclosure; and b) a chloroplast transit peptide. Thus, for example, the CRISPR-CasZ complex can be targeted to the chloroplast. In some cases, this targeting can be achieved by the presence of an N-terminal extension, which is called a chloroplast transit peptide (CTP) or a plastid transit peptide. If the expressed polypeptide is to be compartmentalized in a plant plastid (e.g., a chloroplast), the chromosomal transgene from a bacterial source must have a sequence encoding a CTP sequence, which is fused to a sequence encoding the expressed polypeptide. Therefore, the localization of exogenous polypeptides to chloroplasts is usually achieved by operably linking a polynucleotide sequence encoding a CTP sequence to a 5' region of a polynucleotide encoding an exogenous polypeptide. During translocation to the plastid, CTP is removed in a processing step. However, processing efficiency may be affected by the amino acid sequence of the CTP and the sequence near the NH2 end of the peptide. Other options that have been described for targeting chloroplasts are the maize cab-m7 signal sequence (US Pat. No. 7,022,896, WO97 / 41228), the pea glutathione reductase signal sequence (WO97 / 41228), and the CTP described in US2009029861.

[0123] In some cases, the CasZ fusion polypeptide of the present disclosure may include: a) a CasZ polypeptide of the present disclosure; and b) an endosomal escape peptide. In some cases, the endosomal escape polypeptide comprises the amino acid sequence GLFXALLXLLXSLWXLLLXA (SEQ ID NO: 112), wherein each X is independently selected from lysine, histidine, and arginine. In some cases, the endosomal escape polypeptide comprises the amino acid sequence GLFHALLHLLHSLWHLLLHA (SEQ ID NO: 113).

[0124] 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 regulation and / or target protein modification, such as 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 USA. 2009 Mar 31;106(13):5053-8; Akopian et al., Proc Natl Acad Sci USA. 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 USA. 2003 Oct 14;100(21):11997-2002; Papworth et al., Proc Natl Acad Sci USA. 2009 Mar 31;106(13):5053-8; Akopian et al., Proc Natl Acad Sci USA. 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 USA. 2003 Oct 14;100(21):11997-2002; Papworth et al., Proc Natl Acad Sci USA. USA. 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 USA. 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.2016 Jun 23; 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;23(10):1163-71. Month;10(10):977-9. .

[0125] In addition, suitable heterologous polypeptides include, but are not limited to, polypeptides that directly and / or indirectly provide increased transcription and / or translation of target nucleic acids (e.g., transcription activators or fragments thereof, proteins or fragments thereof that recruit transcription activators, small molecules / drug responsive transcription and / or translation regulatory factors, translation regulatory proteins, etc.). Non-limiting examples of heterologous polypeptides that achieve increased or decreased transcription include transcription activator domains and transcription repressor domains. In some such cases, the chimeric CasZ polypeptide is targeted to a specific position (i.e., sequence) in the target nucleic acid by a guide nucleic acid (guide RNA) and plays a role in locus-specific regulation, such as blocking RNA polymerase and promoter (the promoter selectively inhibits transcription activator function) binding and / or modifying local chromatin state (e.g., when using a fusion sequence, modifying the target nucleic acid or modifying a polypeptide associated with the target nucleic acid). In some cases, the change is transient (e.g., transcription repression or activation). In some cases, the change is heritable (e.g., when epigenetic modification is performed on a target nucleic acid or a protein associated with a target nucleic acid (e.g., nucleosome histones).

[0126] When targeting ssRNA target nucleic acids, non-limiting examples of heterologous polypeptides used 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 acting on RNA (ADARs), including A to I and / or C to U editing enzymes); helicases; RNA binding proteins, etc. It should be understood that the heterologous polypeptide may include an entire protein, or in some cases, may include a fragment (e.g., a functional domain) of a protein.

[0127] The heterologous polypeptide of the subject chimeric CasZ polypeptide can be any domain capable of interacting with ssRNA (for purposes of this disclosure, which includes intramolecular and / or intermolecular secondary structures, e.g., double-stranded RNA duplexes, such as hairpins, stem-loops, etc.), whether transiently or irreversibly, directly or indirectly, including, but not limited to, an effector domain selected from the group consisting of: endonucleases (e.g., RNase III, CRR22DYW domain, Dicer and PIN (PilTN terminal) domains from proteins such as SMG5 and SMG6); proteins and protein domains responsible for stimulating RNA cleavage (e.g., CPSF, CstF, CFIm, and CFIIm); exonucleases (e.g., XRN-1 or exonuclease T); deadenylases (e.g., HNT3); proteins and protein domains responsible for nonsense-mediated RNA decay (e.g., UPF1, UPF2, UPF3, UPF3b, RNPS1, 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) regulating 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 uridylate transferase); proteins and protein domains responsible for RNA localization (e.g., for example, 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 hnRNPA1); proteins and protein domains responsible for stimulating RNA splicing (e.g., serine / arginine-rich (SR) domains); proteins and protein domains responsible for reducing transcription efficiency (e.g., FUS (TLS)); and proteins and protein domains responsible for stimulating transcription (e.g., CDK7 and HIVTat).Alternatively, the effector domain may be selected from the group comprising: endonucleases; proteins and protein domains capable of stimulating RNA cleavage; exonucleases; deadenylases; proteins and protein domains having 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 regulating translation (e.g., translation factors, such as initiation factors, elongation factors, release factors, etc., e.g., eIF4G); proteins and protein domains capable of polyadenylation of RNA; proteins and protein domains capable of polyuridylation of RNA; proteins and protein domains having RNA localization activity; proteins and protein domains capable of nuclear retention of RNA; proteins and protein domains having 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 reducing transcription efficiency; and proteins and protein domains capable of stimulating transcription. Another suitable heterologous polypeptide is the PUF RNA binding domain, which is described in more detail in WO2012068627, which is herein incorporated by reference in its entirety.

[0128] Some RNA splicing factors that can be used as heterologous polypeptides (whole or as fragments thereof) of chimeric CasZ polypeptides have a modular structure 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 an exon splicing enhancer (ESE) in the pre-mRNA and a C-terminal RS domain that promotes exon inclusion. As another example, the hnRNP protein hnRNPAl binds to an exon splicing silencer (ESS) through its RRM domain and inhibits exon inclusion through a C-terminal glycine-rich domain. Some splicing factors can regulate the alternative use of splice sites (ss) by binding to a regulatory sequence between two alternative sites. For example, ASF / SF2 can recognize ESE and facilitate the use of intron proximal sites, while hnRNPAl can bind to ESS and divert splicing to the use of intron distal sites. One application of such factors is to generate ESFs that regulate alternative splicing of endogenous genes, particularly disease-related genes. For example, Bcl-x pre-mRNA produces two splice isoforms that have two alternative 5' splice sites to encode 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, thereby 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 (e.g., developing lymphocytes). The ratio of the two Bcl-x splice isoforms is determined by a number of splice sites located in the core exon region or the exon extension region (i.e., between the two alternative 5' splice sites). Element regulation. For more examples, see WO2010075303, which is hereby incorporated by reference in its entirety.

[0129] Additional suitable fusion partners include, but are not limited to, proteins (or fragments thereof) that act as boundary elements (e.g., CTCF), proteins and fragments thereof that provide peripheral recruitment (e.g., lamin A, lamin B, etc.), protein docking elements (e.g., FKBP / FRB, Pil1 / Aby1, etc.).

[0130] Examples of various additional suitable heterologous polypeptides (or fragments thereof) for use with the subject chimeric CasZ polypeptides include, but are not limited to, those described in the following applications (the publications relate to other CRISPR endonucleases such as Cas9, but the described fusion partners may also be used with CasZ): PCT patent applications: WO2010075303, WO2012068627, and WO2013155555, and can be found, for example, in 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; said patents are hereby incorporated by reference in their entirety.

[0131] In some cases, the 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 keeping 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 embodiments, the CasZ fusion polypeptide does not include an NLS, so that the protein is not targeted to the nucleus (which may be advantageous, for example, when the target nucleic acid is an RNA present in the cytosol). In some embodiments, the heterologous polypeptide can provide a tag (i.e., the heterologous polypeptide is a detectable marker) that facilitates tracking and / or purification (e.g., a fluorescent protein, such as green fluorescent protein (GFP), YFP, RFP, CFP, mCherry, tdTomato, etc.; a histidine tag, such as a 6XHis tag; a hemagglutinin (HA) tag; a FLAG tag; a Myc tag, etc.).

[0132] In some cases, the CasZ protein (e.g., a wild-type CasZ protein, a variant CasZ protein, a chimeric CasZ protein, a dCasZ protein, a chimeric CasZ protein in which the CasZ portion has reduced nuclease activity-such as a dCasZ protein fused to a fusion partner, etc.) comprises (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, the CasZ polypeptide comprises one or more NLSs (e.g., 2 or more, 3 or more, 4 or more, or 5 or more NLSs). In some cases, one or more NLSs (2 or more, 3 or more, 4 or more, or 5 or more NLSs) are positioned at or near the N-terminus and / or C-terminus (e.g., within 50 amino acids). In some cases, one or more NLSs (2 or more, 3 or more, 4 or more, or 5 or more NLSs) are positioned at or near the N-terminus (e.g., within 50 amino acids). In some cases, one or more NLS (2 or more, 3 or more, 4 or more, or 5 or more NLS) are located at or near the C-terminus (e.g., within 50 amino acids). In some cases, one or more NLS (3 or more, 4 or more, or 5 or more NLS) are located at or near both the N-terminus and the C-terminus (e.g., within 50 amino acids). In some cases, the NLS is located at the N-terminus, and the NLS is located at the C-terminus.

[0133] In some cases, the CasZ protein (e.g., a wild-type CasZ protein, a variant CasZ protein, a chimeric CasZ protein, a dCasZ protein, a chimeric CasZ protein wherein the CasZ portion has reduced nuclease activity, such as a dCasZ protein fused to a fusion partner, 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, the CasZ protein (e.g., a wild-type CasZ protein, a variant CasZ protein, a chimeric CasZ protein, a dCasZ protein, a chimeric CasZ protein wherein the CasZ portion has reduced nuclease activity, such as a dCasZ protein fused to a fusion partner, etc.) comprises (is fused to) between 2 and 5 NLSs (e.g., 2-4 or 2-3 NLSs).

[0134] 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: 114); an NLS from a nucleoplasmin (e.g., a nucleoplasmin bipartite NLS having the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 115)); a c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 116) or RQRRNELKRSP (SEQ ID NO: 117); an hRNPA1M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGG QYFAKPRNQGGY (SEQ ID NO: 118); the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 119) from the IBB domain of importin-α; the sequence VSRKRPRP (SEQ ID NO: 120) of myoma T protein. O:120) and PPKKARED (SEQ ID NO:121); the sequence of human p53 PQPKKKPL (SEQ ID NO:122); the sequence of mouse c-abl IV SALIKKKKKMAP (SEQ ID NO:123); the sequence of influenza virus NS1 DRLRR (SEQ ID NO:124) and PKQ KKRK (SEQ ID NO:125); the sequence of hepatitis virus delta antigen RKLKKKIKKL (SEQ ID NO:126); the sequence of mouse Mx1 protein REKKKFLKRR (SEQ ID NO:127); the sequence of human poly (ADP-ribose) polymerase KRKGDEVDGVDEVAKKKSK K (SEQ ID NO:128); and the sequence of steroid hormone receptor (human) glucocorticoid RKCLQAGMNLEARKTKK (SEQ ID NO:129). Generally speaking, NLS (or multiple NLS) has sufficient strength to drive CasZ protein to accumulate in the nucleus of eukaryotic cells in a detectable amount. Detection of accumulation in the nucleus can be performed by any suitable technique. For example, a detectable marker can be fused to the CasZ protein so that the position in the cell can be visualized. The nucleus can also be isolated from the cell, and then the contents of the nucleus can be analyzed by any suitable method for detecting protein (such as immunohistochemistry, Western blot or enzyme activity assay). Accumulation in the nucleus can also be determined indirectly.

[0135] In some cases, the CasZ fusion polypeptide comprises a "protein transduction domain" or PTD (also known as CPP-cell penetrating peptide), which refers to a polypeptide, polynucleotide, carbohydrate, or organic or inorganic compound that facilitates traversal of a lipid bilayer, a micelle, a cell membrane, an organelle membrane, or a vesicle membrane. The PTD attached to another molecule (which may range from a small polar molecule to a large macromolecule and / or nanoparticle) facilitates the molecule to traverse the membrane, for example, from the extracellular space into the intracellular space or from the cytosol into an organelle. In some embodiments, the PTD is covalently linked to the amino terminus of the polypeptide (e.g., linked to wild-type CasZ to generate a fusion protein, or linked to a variant CasZ protein (such as dCasZ, nickase CasZ, or chimeric CasX protein) to generate a fusion protein). In some embodiments, the PTD is covalently linked to the carboxyl terminus of the polypeptide (e.g., linked to wild-type CasZ to generate a fusion protein, or linked to a variant CasZ protein (such as dCasZ, nickase CasZ, or chimeric CasZ protein) to generate a fusion protein). In some cases, the PTD is inserted into the CasZ fusion polypeptide at a suitable insertion site (i.e., not at the N-terminus or C-terminus of the CasZ fusion polypeptide). In some cases, the subject CasZ fusion polypeptide comprises (is conjugated to, fused to) one or more PTDs (e.g., two or more, three or more, four or more PTDs). In some cases, the PTD comprises a nuclear localization signal (NLS) (e.g., in some cases, 2 or more, 3 or more, 4 or more or 5 or more NLS). Therefore, in some cases, the CasZ fusion 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 PTD is covalently linked to a nucleic acid (e.g., a CasZ guide nucleic acid, a polynucleotide encoding a CasZ guide nucleic acid, a polynucleotide encoding a CasZ fusion polypeptide, a donor polynucleotide, etc.).Examples of PTDs include, but are not limited to, a minimal eleven amino acid polypeptide protein transduction domain (corresponding to residues 47-57 of HIV-1 TAT comprising YGRKKRRQRRR; SEQ ID NO: 130); a polyarginine sequence comprising a sufficient number of arginines (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10-50 arginines) to directly enter a cell; a VP22 domain (Zender et al. (2002) Cancer Gene Ther. 9(6):489-96); Drosophila Antennapedia protein transduction domain (Noguchi et al. (2003) Diabetes 52(7):1732-1737); truncated human calcitonin peptide (Trehin et al. (2004) Pharm. Research 21:1248-1256); polylysine (Wender et al. (2000) Proc. Natl. Acad. Sci. USA 97:13003-13008); RRQRRTSKLMKR (SEQ ID NO:131); transportan GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO:132); KALAWEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO: 133); and RQIKIWFQNRRMKWKK (SEQ ID NO: 134). Exemplary PTDs include, but are not limited to, YGRKKRRQRRR (SEQ ID NO: 130); RKKRRQRRR (SEQ ID NO: 135); arginine homopolymers with 3 to 50 arginine residues; exemplary PTD domain amino acid sequences include, but are not limited to, any one of the following sequences: YGRKKRRQRRR (SEQ ID NO: 130); RKKRRQRR (SEQ ID NO: 136); YARAAARQARA (SEQ ID NO: 137); THRLPRRRRRR (SEQ ID NO: 138); and GGRRARRRRRR (SEQ ID NO: 139). In some embodiments, the PTD is an activatable CPP (ACPP) (Aguilera et al. (2009) Integr Biol (Camb) June; 1(5-6): 371-381). ACPPs include a polycationic CPP (e.g., Arg9 or "R9") linked to a matching polyanion (e.g., Glu9 or "E9") via a cleavable linker, 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 polyarginine and its inherent adhesive properties, thereby "activating" the ACPP to traverse the membrane.

[0136] Linkers (e.g., for fusion partners)

[0137] In some cases, the subject CasZ protein is fused to the fusion partner via a linker polypeptide (e.g., one or more linker polypeptides). The linker polypeptide may have any of a variety of amino acid sequences. The protein may be connected by a spacer peptide, which generally has a flexible nature, but other chemical bonds are not excluded. Suitable linkers include polypeptides having a length between 4 amino acids and 40 amino acids or a length between 4 amino acids and 25 amino acids. These linkers may be produced by using synthetic oligonucleotides encoding linkers to couple proteins, or may be encoded by a nucleic acid sequence encoding a fusion protein. Peptide linkers with a certain degree of flexibility may be used. The connecting peptide may actually have any amino acid sequence, and it should be remembered that a preferred linker will have a sequence that produces a generally flexible peptide. The use of small amino acids (such as glycine and alanine) is used to produce flexible peptides. For those skilled in the art, it is conventional to produce such sequences. A variety of different linkers are commercially available and are considered suitable for use.

[0138] Examples of linker polypeptides include glycine polymers (G) n , glycine-serine polymers (including, for example, (GS) n 、GSGGS n (SEQ ID NO: 140), GGSGGS n (SEQ ID NO: 141) and GGGS n (SEQ ID NO: 142), wherein n is an integer of at least 1), glycine-alanine polymer, alanine-serine polymer. Exemplary linkers may comprise amino acid sequences including, but not limited to, GGSG (SEQ ID NO: 143), GGSGG (SEQ ID NO: 144), GSGSG (SEQ ID NO: 145), GSGGG (SEQ ID NO: 146), GGGSG (SEQ ID NO: 147), GSSSG (SEQ ID NO: 148), etc. One of ordinary skill will recognize that the design of a peptide conjugated to any desired element may include a fully or partially flexible linker, such that the linker may include a flexible linker and one or more portions that confer a less flexible structure.

[0139] Detectable Label

[0140] In some cases, the CasZ polypeptides of the present disclosure comprise (e.g., can be linked / fused to) a detectable label. Suitable detectable labels and / or moieties that can provide a detectable signal may include, but are not limited to, enzymes, radioisotopes, members of specific binding pairs, fluorophores, fluorescent proteins, quantum dots, and the like.

[0141] Suitable fluorescent proteins include, but are not limited to, green fluorescent protein (GFP) or variants thereof, a blue fluorescent variant of GFP (BFP), a cyan fluorescent variant of GFP (CFP), a 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, dimer 2, t-dimer 2 (12), mRFP1, pocilloporin, Renilla GFP, MonsterGFP, paGFP, Kaede protein, and kindling protein. Protein), phycobiliprotein 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), etc. Any of a variety of fluorescent proteins and colored proteins from coral polyp species as described, for example, in Matz et al. (1999) Nature Biotechnol. 17:969-973 is suitable for use.

[0142] Suitable enzymes include, but are not limited to, horseradish peroxidase (HRP), alkaline phosphatase (AP), β-galactosidase (GAL), glucose-6-phosphate dehydrogenase, β-N-acetylglucosaminidase, β-glucuronidase, invertase, xanthine oxidase, firefly luciferase, glucose oxidase (GO), and the like.

[0143] Protospacer adjacent motif (PAM)

[0144] The natural CasZ protein binds to the target DNA at a target sequence defined by a region of complementarity between the DNA-targeting RNA and the target DNA. As with many CRISPR endonucleases, site-specific binding (and / or cleavage) of the double-stranded target DNA occurs at a position determined by: (i) base pairing complementarity between the guide RNA and the target DNA; and (ii) a short motif in the target DNA [called a protospacer adjacent motif (PAM)].

[0145] In some cases, the PAM of the CasZ protein is directly located at the 5' end of the target sequence of the non-complementary strand of the target DNA (also referred to as the non-target strand; the complementary strand hybridizes with the guide sequence of the guide RNA, while the non-complementary strand does not directly hybridize with the guide RNA and is the reverse complement of the non-complementary strand). In some cases (e.g., for CasZc), the PAM sequence of the non-complementary strand is 5'-TTA-3'. In some cases (e.g., for CasZb), the PAM sequence of the non-complementary strand is 5'-TTTN-3'. In some cases (e.g., for CasZb), the PAM sequence of the non-complementary strand is 5'-TTTA-3'.

[0146] In some cases, different CasZ proteins (i.e., CasZ proteins from various species) can be advantageously used in various provided methods to take advantage of various enzymatic features of different CasZ proteins (e.g., for different PAM sequence preferences; for increased or decreased enzymatic activity; for increased or decreased cytotoxicity levels; for changing the balance between NHEJ, homology-directed repair, single-strand breaks, double-strand breaks, etc.; for taking advantage of short total sequences, etc.). CasZ proteins from different species may require different PAM sequences in the target DNA. Therefore, for the specific CasZ protein selected, the PAM sequence preference may be different from one or more of the above sequences. 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 can be used.

[0147] CasZ guide RNA

[0148] Nucleic acid molecules that bind to the CasZ 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 "CasZ guide RNA" or simply "guide RNA". It should be understood that in some cases, hybrid DNA / RNA can be prepared so that the CasZ guide RNA contains DNA bases in addition to RNA bases, but the term "CasZ guide RNA" is still used to cover such molecules herein.

[0149] It can be said that the CasZ guide RNA comprises two segments, namely a targeting segment and a protein binding segment. The targeting segment of the CasZ guide RNA comprises a nucleotide sequence (guide sequence) complementary to (and thus hybridizing with) a specific sequence (target site) within a target nucleic acid (e.g., target ssRNA, target ssDNA, complementary strand of double-stranded target DNA, etc.). The protein binding segment (or "protein binding sequence") interacts (binds) with the CasZ polypeptide. The protein binding segment of the subject CasZ guide RNA comprises two sections of complementary nucleotides that hybridize to form a double-stranded RNA duplex (dsRNA duplex). Site-specific binding and / or cleavage of a target nucleic acid (e.g., genomic DNA) may occur at a position (e.g., a target sequence of a target locus) determined by base pairing complementarity between the CasZ guide RNA (the guide sequence of the CasZ guide RNA) and the target nucleic acid.

[0150] CasZ guide RNA and CasZ protein (e.g., fused CasZ polypeptide) form a complex (e.g., bound by non-covalent interactions). CasZ guide RNA provides target specificity for the complex by comprising a targeting segment, wherein the targeting segment comprises a guide sequence (a nucleotide sequence complementary to the target nucleic acid sequence). The CasZ protein of the complex provides site-specific activity (e.g., cleavage activity provided by the CasZ protein and / or activity provided by a fusion partner in the case of a chimeric CasZ protein). In other words, the CasZ protein is directed to a target nucleic acid sequence (e.g., a target sequence) due to its association with the CasZ guide RNA.

[0151] The "guide sequence", also referred to as the "targeting sequence" of the CasZ guide RNA, can be modified so that the CasZ guide RNA can target a CasZ protein (e.g., a naturally occurring CasZ protein, a fusion CasZ polypeptide (chimeric CasZ), etc.) to any desired sequence of any desired target nucleic acid, except that (e.g., as described herein) a PAM sequence can be considered. Thus, for example, a CasZ 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.

[0152] In some cases, the CasZ guide RNA has a length of 30 nucleotides (nt) or more (e.g., 35 nt or more, 40 nt or more, 45 nt or more, 50 nt or more, 55 nt or more, or 60 nt or more). In some embodiments, the CasZ guide RNA has a length of 40 nucleotides (nt) or more (e.g., 45 nt or more, 50 nt or more, 55 nt or more, or 60 nt or more). In some cases, the CasZ guide RNA has a length of 30 nucleotides (nt) to 100 nt (e.g., 30-90, 30-80, 30-75, 30-70, 30-65, 40-100, 40-90, 40-80, 40-75, 40-70, or 40-65 nt). In some cases, the CasZ guide RNA has a length of 40 nucleotides (nt) to 100 nt (e.g., 40-90, 40-80, 40-75, 40-70, or 40-65 nt).

[0153] Guide sequence of CasZ guide RNA

[0154] The subject CasZ guide RNA comprises a guide sequence (ie, a targeting sequence), which is a nucleotide sequence complementary to a sequence in a target nucleic acid (target site). In other words, the guide sequence of the CasZ guide RNA can interact with a target nucleic acid (eg, double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), single-stranded RNA (ssRNA), or double-stranded RNA (dsRNA)) in a sequence-specific manner by hybridization (ie, base pairing). The guide sequence of the CasZ guide RNA can be modified (eg, by genetic engineering) / designed to hybridize with any desired target sequence within a target nucleic acid (eg, a eukaryotic target nucleic acid, such as genomic DNA) (eg, when considering a PAM, eg, when targeting a dsDNA target).

[0155] 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%.

[0156] 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 at the 3'-most end of the target site of the target nucleic acid.

[0157] 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., 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 17 or more (e.g., 18 or more, 19 or more, 20 or more, 21 or more, 22 or more) consecutive 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 17 or more (e.g., 18 or more, 19 or more, 20 or more, 21 or more, 22 or more) consecutive 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 17 or more (e.g., 18 or more, 19 or more, 20 or more, 21 or more, 22 or more) consecutive nucleotides. In some cases, the percent 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.

[0158] 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., 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 or more (e.g., 20 or more, 21 or more, 22 or more) consecutive 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 or more (e.g., 20 or more, 21 or more, 22 or more) consecutive 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 or more (e.g., 20 or more, 21 or more, 22 or more) consecutive 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) consecutive nucleotides.

[0159] 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 17-25 consecutive 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 17-25 consecutive 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 17-25 consecutive 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 consecutive nucleotides.

[0160] 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 consecutive 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 consecutive 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 consecutive 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 consecutive nucleotides.

[0161] 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.

[0162] Protein-binding segment of CasZ guide RNA

[0163] The protein binding segment of the subject CasZ guide RNA interacts with the CasZ protein. The CasZ guide RNA guides the bound CasZ protein to a specific nucleotide sequence in the target nucleic acid through the guide sequence mentioned above. The protein binding segment of the CasZ guide RNA comprises two sections of nucleotides, which are complementary to each other and hybridize to form a double-stranded RNA duplex (dsRNA duplex). Therefore, the protein binding segment comprises a dsRNA duplex.

[0164] 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 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, such as 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 in the duplex region are paired, and therefore the duplex formation region may include a projection. The term "bulge" herein is used to refer to a section of nucleotides (which may be one or more nucleotides) that does not contribute to the double-stranded duplex, but is surrounded by contributing nucleotides at the 5' and 3' ends, and thus the bulge is considered to be 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).

[0165] Thus, in some cases, the nucleotide segments 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 nucleotide segments 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 nucleotide segments 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 nucleotide segments that hybridize to each other to form a dsRNA duplex are 70%-95% complementary to each other (e.g., 75%-95%, 80%-95%, 85%-95%, 90%-95% complementary).

[0166] In other words, in some embodiments, the dsRNA duplex comprises two stretches of nucleotides that are 70%-100% complementary to each other (e.g., 75%-100%, 80%-10%, 85%-100%, 90%-100%, 95%-100% complementary). In some cases, the dsRNA duplex comprises two stretches of nucleotides that are 85%-100% complementary to each other (e.g., 90%-100%, 95%-100% complementary). In some cases, the dsRNA duplex comprises two stretches of nucleotides that are 70%-95% complementary to each other (e.g., 75%-95%, 80%-95%, 85%-95%, 90%-95% complementary).

[0167] The duplex region of the subject CasZ guide RNA may comprise one or more (1, 2, 3, 4, 5, etc.) mutations relative to a naturally occurring duplex region. For example, in some cases, base pairs may be maintained while the nucleotides contributing to the base pairs of each segment may be different. In some cases, the duplex region of the subject CasZ guide RNA comprises more paired bases, fewer paired bases, smaller bulges, larger bulges, fewer bulges, more bulges, or any convenient combination thereof compared to a naturally occurring duplex region (of a naturally occurring CasZ guide RNA).

[0168] Examples of various Cas9 guide RNAs and cpf1 guide RNAs can be found in the art, and in some cases, modifications similar to those introduced into the Cas9 guide RNA can also be introduced into the CasZ guide RNA of the present disclosure (e.g., mutations in the dsRNA duplex region, extensions at the 5' or 3' end for increased stability to provide interaction 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 USA. 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 Feb 28;152(5):1173-83; Wang et al., Proc Natl Acad Sci USA. 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 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. Acids Res. 2013 Nov 1;41(20):e187; Hu et al., Cell Res. 2013 Nov 1;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 USA. 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.2014 Oct 23;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; all of which are hereby incorporated by reference in their entirety. .

[0169] The CasZ guide RNA comprises a guide sequence and two segments of nucleotides ("duplex-forming segments") that hybridize to form a dsRNA duplex of a protein-binding segment. The specific sequence of a given CasZ guide RNA may be characteristic of the species in which the crRNA is found. Examples of suitable CasZ guide RNAs are provided herein.

[0170] Exemplary guide RNA sequences

[0171] The repeat sequences of crRNAs of naturally occurring CasZ proteins (non-guide sequence portions of CasZ guide RNAs) are shown in Tables 1 and 3 (see, e.g., Figure 1 and Figure 7 ).

[0172] Table 1. crRNA repeat sequences of CasZ proteins

[0173]

[0174]

[0175] In some cases, the subject CasZ guide RNA comprises (e.g., in addition to a guide sequence, e.g., as part of a protein binding region) a crRNA sequence of Table 1 or Table 3. In some cases, the subject CasZ guide RNA comprises a nucleotide sequence having 70% or more identity (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 100% identity) with a crRNA sequence of Table 1 or Table 3. In some cases, the subject CasZ guide RNA comprises a nucleotide sequence having 80% or more identity (e.g., 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 100% identity) with a crRNA sequence of Table 1 or Table 3. In some cases, a subject CasZ guide RNA comprises a nucleotide sequence having 90% or more identity (e.g., 93% or more, 95% or more, 97% or more, 98% or more, or 100% identity) to a crRNA sequence of Table 1 or Table 3.

[0176] In some cases, a subject CasZ guide RNA comprises (e.g., in addition to a guide sequence, e.g., as part of a protein binding region) a CasZa, CasZb, CasZc, CasZd, CasZe, CasZf, CasZg, CasZh, or CasZi crRNA sequence of Table 1 or Table 3. In some cases, a subject CasZ guide RNA comprises a nucleotide sequence having 70% or more identity (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 100% identity) to a CasZa, CasZb, CasZc, CasZd, CasZe, CasZf, CasZg, CasZh, or CasZi crRNA sequence of Table 1 or Table 3. In some cases, the subject CasZ guide RNA comprises a nucleotide sequence having 80% or more identity (e.g., 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 100% identity) to a CasZa, CasZb, CasZc, CasZd, CasZe, CasZf, CasZg, CasZh, or CasZi crRNA sequence of Table 1 or Table 3. In some cases, the subject CasZ guide RNA comprises a nucleotide sequence having 90% or more identity (e.g., 93% or more, 95% or more, 97% or more, 98% or more, or 100% identity) to a CasZa, CasZb, CasZc, CasZd, CasZe, CasZf, CasZg, CasZh, or CasZi crRNA sequence of Table 1 or Table 3.

[0177] In some cases, the subject CasZ guide RNA comprises (e.g., in addition to a guide sequence, e.g., as part of a protein binding region) a CasZa crRNA sequence of Table 1 or Table 3. In some cases, the subject CasZ guide RNA comprises a nucleotide sequence having 70% or more identity (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 100% identity) with a CasZa crRNA sequence of Table 1 or Table 3. In some cases, the subject CasZ guide RNA comprises a nucleotide sequence having 80% or more identity (e.g., 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 100% identity) with a CasZa crRNA sequence of Table 1 or Table 3. In some cases, a subject CasZ guide RNA comprises a nucleotide sequence having 90% or more identity (e.g., 93% or more, 95% or more, 97% or more, 98% or more, or 100% identity) to a CasZa crRNA sequence of Table 1 or Table 3.

[0178] In some cases, the subject CasZ guide RNA comprises (e.g., in addition to a guide sequence, e.g., as part of a protein binding region) a CasZb crRNA sequence of Table 1 or Table 3. In some cases, the subject CasZ guide RNA comprises a nucleotide sequence having 70% or more identity (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 100% identity) with a CasZb crRNA sequence of Table 1 or Table 3. In some cases, the subject CasZ guide RNA comprises a nucleotide sequence having 80% or more identity (e.g., 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 100% identity) with a CasZb crRNA sequence of Table 1 or Table 3. In some cases, a subject CasZ guide RNA comprises a nucleotide sequence having 90% or more identity (e.g., 93% or more, 95% or more, 97% or more, 98% or more, or 100% identity) to a CasZb crRNA sequence of Table 1 or Table 3.

[0179] In some cases, the subject CasZ guide RNA comprises (e.g., in addition to a guide sequence, e.g., as part of a protein binding region) a CasZc crRNA sequence of Table 1 or Table 3. In some cases, the subject CasZ guide RNA comprises a nucleotide sequence having 70% or more identity (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 100% identity) with a CasZc crRNA sequence of Table 1 or Table 3. In some cases, the subject CasZ guide RNA comprises a nucleotide sequence having 80% or more identity (e.g., 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 100% identity) with a CasZc crRNA sequence of Table 1 or Table 3. In some cases, a subject CasZ guide RNA comprises a nucleotide sequence having 90% or more identity (e.g., 93% or more, 95% or more, 97% or more, 98% or more, or 100% identity) to a CasZc crRNA sequence of Table 1 or Table 3.

[0180] In some cases, the subject CasZ guide RNA comprises (e.g., in addition to a guide sequence, e.g., as part of a protein binding region) a CasZd crRNA sequence of Table 1 or Table 3. In some cases, the subject CasZ guide RNA comprises a nucleotide sequence having 70% or more identity (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 100% identity) with a CasZd crRNA sequence of Table 1 or Table 3. In some cases, the subject CasZ guide RNA comprises a nucleotide sequence having 80% or more identity (e.g., 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 100% identity) with a CasZd crRNA sequence of Table 1 or Table 3. In some cases, a subject CasZ guide RNA comprises a nucleotide sequence having 90% or more identity (e.g., 93% or more, 95% or more, 97% or more, 98% or more, or 100% identity) to a CasZd crRNA sequence of Table 1 or Table 3.

[0181] In some cases, the subject CasZ guide RNA comprises (e.g., in addition to a guide sequence, e.g., as part of a protein binding region) a CasZe crRNA sequence of Table 1 or Table 3. In some cases, the subject CasZ guide RNA comprises a nucleotide sequence having 70% or more identity (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 100% identity) with a CasZe crRNA sequence of Table 1 or Table 3. In some cases, the subject CasZ guide RNA comprises a nucleotide sequence having 80% or more identity (e.g., 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 100% identity) with a CasZe crRNA sequence of Table 1 or Table 3. In some cases, a subject CasZ guide RNA comprises a nucleotide sequence having 90% or more identity (e.g., 93% or more, 95% or more, 97% or more, 98% or more, or 100% identity) to a CasZe crRNA sequence of Table 1 or Table 3.

[0182] In some cases, the subject CasZ guide RNA comprises (e.g., excluding a guide sequence, e.g., as part of a protein binding region) a CasZf crRNA sequence of Table 1 or Table 3. In some cases, the subject CasZ guide RNA comprises a nucleotide sequence having 70% or more identity (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 100% identity) with a CasZf crRNA sequence of Table 1 or Table 3. In some cases, the subject CasZ guide RNA comprises a nucleotide sequence having 80% or more identity (e.g., 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 100% identity) with a CasZf crRNA sequence of Table 1 or Table 3. In some cases, a subject CasZ guide RNA comprises a nucleotide sequence having 90% or more identity (e.g., 93% or more, 95% or more, 97% or more, 98% or more, or 100% identity) to a CasZf crRNA sequence of Table 1 or Table 3.

[0183] In some cases, the subject CasZ guide RNA comprises (e.g., in addition to a guide sequence, e.g., as part of a protein binding region) a CasZg crRNA sequence of Table 1 or Table 3. In some cases, the subject CasZ guide RNA comprises a nucleotide sequence having 70% or more identity (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 100% identity) with a CasZg crRNA sequence of Table 1 or Table 3. In some cases, the subject CasZ guide RNA comprises a nucleotide sequence having 80% or more identity (e.g., 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 100% identity) with a CasZg crRNA sequence of Table 1 or Table 3. In some cases, a subject CasZ guide RNA comprises a nucleotide sequence having 90% or more identity (e.g., 93% or more, 95% or more, 97% or more, 98% or more, or 100% identity) to a CasZg crRNA sequence of Table 1 or Table 3.

[0184] In some cases, the subject CasZ guide RNA comprises (e.g., in addition to a guide sequence, e.g., as part of a protein binding region) a CasZh crRNA sequence of Table 1 or Table 3. In some cases, the subject CasZ guide RNA comprises a nucleotide sequence having 70% or more identity (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 100% identity) with a CasZh crRNA sequence of Table 1 or Table 3. In some cases, the subject CasZ guide RNA comprises a nucleotide sequence having 80% or more identity (e.g., 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 100% identity) with a CasZh crRNA sequence of Table 1 or Table 3. In some cases, a subject CasZ guide RNA comprises a nucleotide sequence having 90% or more identity (e.g., 93% or more, 95% or more, 97% or more, 98% or more, or 100% identity) to a CasZh crRNA sequence of Table 1 or Table 3.

[0185] In some cases, the subject CasZ guide RNA comprises (e.g., in addition to a guide sequence, e.g., as part of a protein binding region) a CasZi crRNA sequence of Table 1 or Table 3. In some cases, the subject CasZ guide RNA comprises a nucleotide sequence having 70% or more identity (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 100% identity) with a CasZi crRNA sequence of Table 1 or Table 3. In some cases, the subject CasZ guide RNA comprises a nucleotide sequence having 80% or more identity (e.g., 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 100% identity) with a CasZi crRNA sequence of Table 1 or Table 3. In some cases, a subject CasZ guide RNA comprises a nucleotide sequence having 90% or more identity (e.g., 93% or more, 95% or more, 97% or more, 98% or more, or 100% identity) to a CasZi crRNA sequence of Table 1 or Table 3.

[0186] In some cases, the subject CasZ guide RNA comprises (e.g., in addition to a guide sequence, e.g., as part of a protein binding region) a CasZj crRNA sequence of Table 1 or Table 3. In some cases, the subject CasZ guide RNA comprises a nucleotide sequence having 70% or more identity (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 100% identity) with a CasZj crRNA sequence of Table 1 or Table 3. In some cases, the subject CasZ guide RNA comprises a nucleotide sequence having 80% or more identity (e.g., 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 100% identity) with a CasZj crRNA sequence of Table 1 or Table 3. In some cases, a subject CasZ guide RNA comprises a nucleotide sequence having 90% or more identity (e.g., 93% or more, 95% or more, 97% or more, 98% or more, or 100% identity) to a CasZj crRNA sequence of Table 1 or Table 3.

[0187] In some cases, the subject CasZ guide RNA comprises (e.g., in addition to a guide sequence, e.g., as part of a protein binding region) a CasZk crRNA sequence of Table 1 or Table 3. In some cases, the subject CasZ guide RNA comprises a nucleotide sequence having 70% or more identity (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 100% identity) with a CasZk crRNA sequence of Table 1 or Table 3. In some cases, the subject CasZ guide RNA comprises a nucleotide sequence having 80% or more identity (e.g., 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 100% identity) with a CasZk crRNA sequence of Table 1 or Table 3. In some cases, a subject CasZ guide RNA comprises a nucleotide sequence having 90% or more identity (e.g., 93% or more, 95% or more, 97% or more, 98% or more, or 100% identity) to a CasZk crRNA sequence of Table 1 or Table 3.

[0188] In some cases, the subject CasZ guide RNA comprises (e.g., in addition to a guide sequence, e.g., as part of a protein binding region) a CasZ1 crRNA sequence of Table 1 or Table 3. In some cases, the subject CasZ guide RNA comprises a nucleotide sequence having 70% or more identity (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 100% identity) to a CasZ1 crRNA sequence of Table 1 or Table 3. In some cases, the subject CasZ guide RNA comprises a nucleotide sequence having 80% or more identity (e.g., 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 100% identity) to a CasZ1 crRNA sequence of Table 1 or Table 3. In some cases, a subject CasZ guide RNA comprises a nucleotide sequence having 90% or more identity (e.g., 93% or more, 95% or more, 97% or more, 98% or more, or 100% identity) to a CasZ1 crRNA sequence of Table 1 or Table 3.

[0189] In some cases, the subject CasZ guide RNA comprises (e.g., in addition to a guide sequence, e.g., as part of a protein binding region) a CasZj, CasZl, or CasZk crRNA sequence of Table 1 or Table 3. In some cases, the subject CasZ guide RNA comprises a nucleotide sequence having 70% or more identity (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 100% identity) to a CasZj, CasZl, or CasZk crRNA sequence of Table 1 or Table 3. In some cases, the subject CasZ guide RNA comprises a nucleotide sequence having 80% or more identity (e.g., 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 100% identity) to a CasZj, CasZl, or CasZk crRNA sequence of Table 1 or Table 3. In some cases, a subject CasZ guide RNA comprises a nucleotide sequence having 90% or more identity (e.g., 93% or more, 95% or more, 97% or more, 98% or more, or 100% identity) to a CasZj, CasZl, or CasZk crRNA sequence of Table 1 or Table 3.

[0190] CasZ transactivates noncoding RNA (trancRNA)

[0191] The compositions and methods of the present disclosure include CasZ transactivating noncoding RNA ("trancRNA"; also referred to herein as "CasZ trancRNA"). In some cases, trancRNA forms a complex with the CasZ polypeptide and CasZ guide RNA of the present disclosure. TranscRNA can be identified as a highly transcribed RNA encoded by a nucleotide sequence present in the CasZ locus. The sequence encoding trancRNA is generally located between the cas gene and the array (repeating sequence) of the CasZ locus (e.g., may be located near the repeating sequence). The following examples illustrate the detection of CasZ trancRNA. In some cases, CasZ trancRNA and CasZ polypeptide are co-immunoprecipitated (forming a complex). In some cases, the presence of CasZ trancRNA is required for system function. Data related to trancRNA (e.g., expression of trancRNA and its position on a naturally occurring array) are provided in the Examples section below.

[0192] In some cases, CasZ trancRNA has a length of 60 nucleotides (nt) to 270 nt (e.g., 60-260, 70-270, 70-260 or 75-255 nt). In some cases, CasZ trancRNA (e.g., CasZa trancRNA) has a length of 60-150 nt (e.g., 60-140, 60-130, 65-150, 65-140, 65-130, 70-150, 70-140 or 70-130 nt). In some cases, CasZ trancRNA (e.g., CasZa trancRNA) has a length of 70-130 nt. In some cases, CasZ trancRNA (e.g., CasZa trancRNA) has a length of about 80 nt. In some cases, CasZ trancRNA (e.g., CasZa trancRNA) has a length of about 90 nt. In some cases, CasZ trancRNA (e.g., CasZa trancRNA) has a length of about 120 nt.

[0193] In some cases, CasZ trancRNA (e.g., CasZb trancRNA) has a length of 85-240 nt (e.g., 85-230, 85-220, 85-150, 85-130, 95-240, 95-230, 95-220, 95-150 or 95-130 nt). In some cases, CasZ trancRNA (e.g., CasZb trancRNA) has a length of 95-120 nt. In some cases, CasZ trancRNA (e.g., CasZb trancRNA) has a length of about 105 nt. In some cases, CasZ trancRNA (e.g., CasZb trancRNA) has a length of about 115 nt. In some cases, CasZ trancRNA (e.g., CasZb trancRNA) has a length of about 215 nt.

[0194] In some cases, CasZ trancRNA (e.g., CasZc trancRNA) has a length of 80-275 nt (e.g., 85-260 nt). In some cases, CasZ trancRNA (e.g., CasZc trancRNA) has a length of 80-110 nt (e.g., 85-105 nt). In some cases, CasZ trancRNA (e.g., CasZc trancRNA) has a length of 235-270 nt (e.g., 240-260 nt). In some cases, CasZ trancRNA (e.g., CasZc trancRNA) has a length of about 95 nt. In some cases, CasZ trancRNA (e.g., CasZc trancRNA) has a length of about 250 nt.

[0195] Exemplary trancRNA sequences

[0196] Examples of trancRNA sequences of naturally occurring CasZ proteins are shown in Table 2.

[0197] Table 2. CasZ trancRNA sequences

[0198]

[0199]

[0200] In some cases, the subject CasZ trancRNA comprises the above CasZa trancRNA sequence. In some cases, the subject CasZ trancRNA comprises a nucleotide sequence having 70% or more identity (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 100% identity) with the above CasZa trancRNA sequence. In some cases, the subject CasZ trancRNA comprises a nucleotide sequence having 80% or more identity (e.g., 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 100% identity) with the above CasZa trancRNA sequence. In some cases, the subject CasZ trancRNA comprises a nucleotide sequence having 90% or higher identity (e.g., 93% or higher, 95% or higher, 97% or higher, 98% or higher, or 100% identity) with the above CasZa trancRNA sequence. In some cases, the subject CasZ trancRNA comprises a nucleotide sequence having 80% or higher identity (e.g., 85% or higher, 90% or higher, 93% or higher, 95% or higher, 97% or higher, 98% or higher, or 100% identity) with the above CasZa trancRNA sequence, and has a length of 60-150 nt (e.g., 60-140, 60-130, 65-150, 65-140, 65-130, 70-150, 70-140, or 70-130 nt).

[0201] In some cases, the subject CasZ trancRNA comprises the above CasZb trancRNA sequence. In some cases, the subject CasZ trancRNA comprises a nucleotide sequence having 70% or more identity (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 100% identity) with the above CasZb trancRNA sequence. In some cases, the subject CasZ trancRNA comprises a nucleotide sequence having 80% or more identity (e.g., 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 100% identity) with the above CasZb trancRNA sequence. In some cases, the subject CasZ trancRNA comprises a nucleotide sequence having 90% or more identity (e.g., 93% or more, 95% or more, 97% or more, 98% or more, or 100% identity) with the above CasZb trancRNA sequence. In some cases, the subject CasZ trancRNA comprises a nucleotide sequence having 80% or more identity (e.g., 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 100% identity) with the above CasZb trancRNA sequence, and has a length of 85-240 nt (e.g., 85-230, 85-220, 85-150, 85-130, 95-240, 95-230, 95-220, 95-150, or 95-130 nt).

[0202] In some cases, the subject CasZ trancRNA comprises the above CasZc trancRNA sequence. In some cases, the subject CasZ trancRNA comprises a nucleotide sequence having 70% or more identity (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 100% identity) with the above CasZc trancRNA sequence. In some cases, the subject CasZ trancRNA comprises a nucleotide sequence having 80% or more identity (e.g., 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 100% identity) with the above CasZc trancRNA sequence. In some cases, the subject CasZ trancRNA comprises a nucleotide sequence having 90% or more identity (e.g., 93% or more, 95% or more, 97% or more, 98% or more, or 100% identity) with the above CasZc trancRNA sequence. In some cases, the subject CasZ trancRNA comprises a nucleotide sequence having 80% or more identity (e.g., 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 100% identity) with the above CasZc trancRNA sequence, and has a length of 80-110 nt (e.g., 85-105 nt) or 235-270 nt (e.g., 240-260 nt).

[0203] In some cases, the subject CasZ trancRNA comprises the above CasZa, CasZb or CasZc trancRNA sequence. In some cases, the subject CasZ trancRNA comprises a nucleotide sequence having 70% or more identity (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 100% identity) with the above CasZa, CasZb or CasZc trancRNA sequence. In some cases, the subject CasZ trancRNA comprises a nucleotide sequence having 80% or more identity (e.g., 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 100% identity) with the above CasZa, CasZb or CasZc trancRNA sequence. In some cases, the subject CasZ trancRNA comprises a nucleotide sequence having 90% or more identity (e.g., 93% or more, 95% or more, 97% or more, 98% or more, or 100% identity) with the above CasZa, CasZb, or CasZc trancRNA sequences. In some cases, the subject CasZ trancRNA comprises a nucleotide sequence having 80% or more identity (e.g., 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 100% identity) with the above CasZa, CasZb, or CasZc trancRNA sequences, and has a length of 60 nucleotides (nt) to 270 nt (e.g., 60-260, 70-270, 70-260, or 75-255 nt).

[0204] In some cases, CasZ trancRNA comprises modified nucleotides (e.g., methylated nucleotides). In some cases, CasZ trancRNA comprises one or more of the following: i) base modification or substitution; ii) backbone modification; iii) modified internucleoside bond; and iv) modified sugar moiety. Possible nucleic acid modifications are described below.

[0205] CASZ system

[0206] The present disclosure provides a CasZ system. The CasZ system of the present disclosure may include one or more of the following: (1) CasZ trans-activating non-coding RNA (trancRNA) (referred to herein as "CasZ trancRNA") or nucleic acid encoding CasZ trancRNA (e.g., expression vector); (2) CasZ protein (e.g., wild-type protein, variant, catalytically impaired variant, CasZ fusion protein, etc.) or nucleic acid encoding CasZ protein (e.g., RNA, expression vector, etc.); and (3) CasZ guide RNA (which binds to CasZ protein and provides sequence specificity for CasZ protein, such as guide RNA that can bind to a target sequence of a eukaryotic genome) or nucleic acid encoding CasZ guide RNA) (e.g., expression vector). The CasZ system may include a host cell (e.g., eukaryotic cell, plant cell, mammalian cell, human cell), the host cell including (1), (2) and (3) one or more (in any combination), for example, in some cases, the host cell includes trancRNA and / or nucleic acid encoding trancRNA. In some cases, the CasZ system comprises (e.g., in addition to the above items) a donor template nucleic acid. In some cases, the CasZ system is a system of one or more nucleic acids (e.g., one or more expression vectors encoding any combination of the above items).

[0207] Nucleic Acids

[0208] The present disclosure provides one or more nucleic acids, the one or more nucleic acids comprising one or more of the following: CasZtrancRNA sequence, nucleotide sequence encoding CasZtrancRNA, nucleotide sequence encoding CasZ polypeptide (e.g., wild-type CasZ protein, nickase CasZ protein, dCasZ Protein, chimeric CasZ protein / CasZ fusion protein, etc.), CasZ guide RNA sequence, nucleotide sequence encoding CasZ guide RNA and donor polynucleotide (donor template, donor DNA) sequence. In some cases, the subject nucleic acid (e.g., one or more nucleic acids) is a recombinant expression vector (e.g., plasmid, viral vector, mini-circle DNA, etc.). In some cases, the nucleotide sequence encoding CasZtrancRNA, the nucleotide sequence encoding CasZ protein and / or the nucleotide sequence encoding CasZ guide RNA are operably connected to a promoter (e.g., an inducible promoter), such as a promoter that can be operated in a selected cell type (e.g., prokaryotic cells, eukaryotic cells, plant cells, animal cells, mammalian cells, primate cells, rodent cells, human cells, etc.).

[0209] In some cases, the nucleotide sequence encoding the CasZ polypeptide of the present disclosure is codon optimized. This type of optimization may require mutations in the nucleotide sequence encoding CasZ to simulate the codon preference of the intended host organism or cell when encoding the same protein at the same time. Therefore, the codons may change, but the encoded protein remains unchanged. For example, if the intended target cell is a human cell, a human codon-optimized nucleotide sequence encoding CasZ may be used. As another non-limiting example, if the intended host cell is a mouse cell, a mouse codon-optimized nucleotide sequence encoding CasZ may be generated. As another non-limiting example, if the intended host cell is a plant cell, a plant codon-optimized nucleotide sequence encoding CasZ may be generated. As another non-limiting example, if the intended host cell is an insect cell, an insect codon-optimized nucleotide sequence encoding CasZ may be generated.

[0210] The present disclosure provides one or more recombinant expression vectors, the one or more recombinant expression vectors comprising (in different recombinant expression vectors in some cases, and in the same recombinant expression vector in some cases): CasZtrancRNA sequence, nucleotide sequence encoding CasZtrancRNA, nucleotide sequence encoding CasZ polypeptide (e.g., wild-type CasZ protein, nickase CasZ protein, dCasZ protein, chimeric CasZ protein / CasZ fusion protein, etc.), CasZ guide RNA sequence, nucleotide sequence encoding CasZ guide RNA and donor polynucleotide (donor template, donor DNA) sequence. In some cases, the subject nucleic acid (e.g., one or more nucleic acids) is a recombinant expression vector (e.g., plasmid, viral vector, minicircle DNA, etc.). In some cases, the nucleotide sequence encoding the CasZ trancRNA, the nucleotide sequence encoding the CasZ protein, and / or the nucleotide sequence encoding the CasZ guide RNA is operably linked to a promoter (e.g., an inducible promoter), such as a promoter 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.).

[0211] Suitable expression vectors include viral expression vectors (e.g., viral vectors based on vaccinia virus; polio virus; 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 9:8186, 1998; Flannery et al., PNAS 94:69166921, 1997; Bennett et al., Invest Opthalmol Vis Sci 38:28572863, 1997; Jomary et al., Gene Ther 4:683690, 1997; Rolling et al., Hum Gene Ther 10:641648, 1999; Ali et al., Hum Mol Genet 5:591594, 1996; Srivastava's WO 93 / 09239; Samulski et al., J. Vir. (1989) 63:3822-3828; Mendelson et al., Virol. (1988) 166:154-165; and Flotte et al. et al., PNAS (1993) 90: 10613-10617); SV40; herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshi et al., PNAS 94: 1031923, 1997; Takahashi et al., J Virol 73: 78127816, 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 leukemia virus, lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus), etc. 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 lentiviral vector. In some cases, the recombinant expression vector of the present disclosure is a recombinant retroviral vector.

[0212] Depending on the host / vector system utilized, any of a number of suitable transcription and translation control elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, and the like may be used in the expression vector.

[0213] In some embodiments, the nucleotide sequence encoding the CasZ guide RNA is operably linked to a control element, e.g., a transcriptional control element, such as a promoter. In some embodiments, the nucleotide sequence encoding the CasZ protein or CasZ fusion polypeptide is operably linked to a control element, e.g., a transcriptional control element, such as a promoter.

[0214] 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., a promoter) is functional in the targeted cell type or the 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, etc.)).

[0215] Non-limiting examples of eukaryotic promoters (promoters that are functional in eukaryotic cells) include EF1α, those promoters from cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, early and late SV40, retroviral long terminal repeats (LTR), and mouse metallothionein-I. The selection of appropriate vectors and promoters is entirely within the level of those of ordinary skill in the art. The expression vector may also contain a ribosome binding site and a transcription terminator for translation initiation. The expression vector may also include a suitable sequence for amplifying expression. The expression vector may also include a nucleotide sequence encoding a protein tag (e.g., a 6xHis tag, a hemagglutinin tag, a fluorescent protein, etc.), which may be fused to the CasZ protein to produce a chimeric CasZ polypeptide.

[0216] In some cases, the nucleotide sequence encoding the CasZ guide RNA and / or the CasZ fusion polypeptide is operably linked to an inducible promoter. In some embodiments, the nucleotide sequence encoding the CasZ guide RNA and / or the CasZ fusion protein is operably linked to a constitutive promoter.

[0217] A promoter can be a constitutively active promoter (i.e., a promoter that is constitutively in the 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 specific temperature, compound, or protein), it can be a spatially restricted promoter (i.e., transcriptional control elements, enhancers, etc.) (e.g., tissue-specific promoters, cell type-specific promoters, etc.), and it can be a temporally restricted promoter (i.e., the promoter is in the "ON" state or the "OFF" state during a specific stage of embryonic development or during a specific stage of a biological process (e.g., the hair follicle cycle in mice)).

[0218] Suitable promoters may be derived from viruses and may therefore be referred to as viral promoters, or they may be derived from any organism, including prokaryotes or eukaryotes. Suitable promoters may 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, SV40 early promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter; adenovirus major late promoter (Ad MLP); herpes simplex virus (HSV) promoter, cytomegalovirus (CMV) promoter such as CMV immediate early promoter region (CMVIE), Rous sarcoma virus (RSV) promoter, human U6 small nuclear promoter (U6) (Miyagishi et al., Nature Biotechnology 20, 497-500 (2002)), enhanced U6 promoter (e.g., Xia et al., Nucleic Acids Res. 2003 September 1; 31 (17)), human H1 promoter (H1), etc.

[0219] In some cases, the nucleotide sequence encoding the CasZ guide RNA is operably linked to (controlled by) a promoter operable in a eukaryotic cell (e.g., U6 promoter, enhanced U6 promoter, H1 promoter, etc.). As understood by those of ordinary skill in the art, when RNA (e.g., guide RNA) is expressed by a nucleic acid (e.g., expression vector) using a U6 promoter (e.g., in a eukaryotic cell) or another PolIII promoter, if there are several Ts (encoding U in RNA) in succession, the RNA may need to be mutated. This is because a string of Ts (e.g., 5 Ts) in DNA can serve as a terminator for polymerase III (PolIII). Therefore, in order to ensure the transcription of the guide RNA in eukaryotic cells, it may sometimes be necessary to modify the sequence encoding the guide RNA to eliminate the effect of T. In some cases, a nucleotide sequence encoding a CasZ protein (e.g., a wild-type CasZ protein, a nickase CasZ protein, a dCasZ protein, a chimeric CasZ protein, etc.) is operably linked to a promoter operable in eukaryotic cells (e.g., a CMV promoter, an EF1α promoter, an estrogen receptor-regulated promoter, etc.).

[0220] Examples of inducible promoters include, but are not limited to, T7 RNA polymerase promoter, T3 RNA polymerase promoter, isopropyl-β-D-thiogalactoside (IPTG)-regulated promoter, lactose-induced promoter, heat shock promoter, tetracycline-regulated promoter, steroid-regulated promoter, metal-regulated promoter, estrogen receptor-regulated promoter, etc. Thus, inducible promoters can be regulated by molecules including, but not limited to, doxycycline; estrogen and / or estrogen analogs; IPTG, etc.

[0221] Inducible promoters suitable for use include any inducible promoters described herein or known to those of ordinary skill in the art. Examples of inducible promoters include, but are not limited to, chemical / 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, including tetracycline repressor protein (tetR), tetracycline operating sequence (tetO) and tetracycline transactivator fusion protein (tTA)), steroid-regulated promoters (e.g., based on rat glucocorticoid receptor, human estrogen receptor, moth hormone receptor, thymidine ... In some embodiments, the promoters of the promoters are regulated by the promoters of the steroid / retinoid / thyroid receptor superfamily, the promoters of the ecdysone receptor, the promoters of the steroid / retinoid / thyroid receptor superfamily, the promoters of the metal-regulated promoters (e.g., promoters derived from metallothionein (proteins that bind and chelate metal ions) genes from yeast, mouse, and human), the promoters of the pathogen-regulated promoters (e.g., promoters inducible by salicylic acid, ethylene, or benzothiadiazole (BTH)), the promoters of the temperature / heat-inducible promoters (e.g., heat shock promoters), and the promoters of the light-regulated promoters (e.g., light-responsive promoters from plant cells).

[0222] 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 specific subset of cells. Spatially restricted promoters may also be referred to as enhancers, transcriptional control elements, control sequences, etc. Any convenient spatially restricted promoter may be used, so long as the promoter is functional in the target host cell (e.g., a eukaryotic cell; a prokaryotic cell).

[0223] 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 separated from and derived from many organisms, such as eukaryotes and prokaryotes. The modification of a reversible promoter derived from a first organism (e.g., a first prokaryote and a second eukaryote, a first eukaryote and a second prokaryote, etc.) for a second organism is well known in the art. Such reversible promoters and systems based on such reversible promoters but also comprising additional control proteins include but are not limited to alcohol-regulated promoters (e.g., alcohol dehydrogenase I (alcA) gene promoter, promoters in response to alcohol transactivator protein (AlcR), etc.), tetracycline-regulated promoters (e.g., promoter systems including Tet activators, TetON, TetOFF, etc.), steroid-regulated promoters (e.g., rat glucocorticoid receptor promoter system, human estrogen receptor promoter system, retinoids Promoter system, thyroid promoter system, ecdysone promoter system, mifepristone promoter system, etc.), metal-regulated promoters (for example, metallothionein promoter system, etc.), pathogen-related regulated promoters (for example, salicylic acid-regulated promoters, ethylene-regulated promoters, benzothiadiazole-regulated promoters, etc.), temperature-regulated promoters (for example, heat shock-inducible promoters (for example, HSP-70, HSP-90, soybean heat shock promoter, etc.)), light-regulated promoters, synthetic inducible promoters, etc.

[0224] Methods for introducing nucleic acids (e.g., DNA or RNA) (e.g., nucleic acids comprising donor polynucleotide sequences, one or more nucleic acids encoding CasZ proteins and / or CasZ guide RNAs and / or CasZ trancRNAs, 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, liposome transfection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran-mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery, etc.

[0225] The introduction of the recombinant expression vector into the cell can occur in any culture medium and under any culture conditions that promote cell survival. The introduction of the recombinant expression vector into the target cell can be performed in vivo or in vitro. The introduction of the recombinant expression vector into the target cell can be performed in vitro.

[0226] In some cases, the CasZ protein can be provided as RNA. RNA can be provided by direct chemical synthesis, or can be transcribed in vitro from DNA (e.g., DNA encoding the CasZ protein). Once synthesized, RNA can be introduced into cells by any well-known technique for introducing nucleic acids into cells (e.g., microinjection, electroporation, transfection, etc.).

[0227] Well-developed transfection techniques can be used (see, e.g., Angel and Yanik (2010) PLoS ONE 5(7):e11756); and commercially available from Qiagen Reagents, Stemfect commercially available from Stemgent TM RNA transfection kits and commercially available from Mirus Bio LLC - mRNA transfection kits provide nucleic acid to cells. See also Beumer et al. (2008) PNAS 105(50): 19821-19826.

[0228] The vector can be provided directly to the target host cell. In other words, the cell is contacted with a vector (e.g., a recombinant expression vector having a donor template sequence and encoding a CasZ guide RNA; a recombinant expression vector encoding a CasZ protein, etc.) comprising the subject nucleic acid so that the vector is absorbed 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.

[0229] Retroviruses, such as lentiviruses, are suitable for the methods of the present disclosure. Commonly used retroviral vectors are "defective", that is, they cannot produce the viral proteins required for productive infection. Moreover, the replication of the vector requires growth in a packaging cell line. In order to generate viral particles containing the target nucleic acid, the retroviral nucleic acid containing the nucleic acid is packaged into a viral capsid by a packaging cell line. Different packaging cell lines provide different envelope proteins (tropic, amphophilic or heterophilic) to be incorporated into the capsid, which determine the specificity of the viral particles to the cell (tropic to mice and rats; amphophilic to most mammalian cell types including humans, dogs and mice; and heterophilic to most mammalian cell types except mouse cells). Appropriate packaging cell lines can be used to ensure that cells are targeted by packaged viral particles. Methods for introducing the subject vector expression vector into the 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).

[0230] The vector for providing the nucleic acid encoding CasZ guide RNA and / or CasZ polypeptide to the target host cell may include a suitable promoter for driving the expression (i.e., transcriptional activation) of the target nucleic acid. In other words, in some cases, the target nucleic acid will be operably connected to the promoter. The promoter may include a ubiquitous activating promoter, such as a CMV-β-actin promoter; or an inducible promoter, such as a promoter that is active in a specific cell population or that responds to the presence of a drug (such as tetracycline). Through transcriptional activation, it is expected that transcription will increase by 10 times, 100 times, and more generally 1000 times in the target cell compared to the basal level. In addition, the vector for providing the nucleic acid encoding CasZ guide RNA and / or CasZ protein to the cell may include the following nucleic acid sequence, which encodes a selectable marker in the target cell to identify cells that have absorbed CasZ guide RNA and / or CasZ protein.

[0231] The nucleic acid comprising the nucleotide sequence encoding the CasZ polypeptide or CasZ fusion polypeptide is RNA in some cases. Therefore, the CasZ fusion protein can be introduced into the cell in the form of RNA. Methods for introducing RNA into cells are known in the art and may include, for example, direct injection, transfection, or any other method for introducing DNA. The CasZ protein may alternatively be provided to the cell in the form of a polypeptide. This polypeptide may optionally be fused to a polypeptide domain that increases the solubility of the product. The domain may be connected to the polypeptide by a defined protease cleavage site (e.g., a TEV sequence cleaved by a TEV protease). The linker may also include one or more flexible sequences, such as 1 to 10 glycine residues. In some embodiments, the cleavage of the fusion protein is performed in a buffer that maintains the solubility of the product, such as in the presence of 0.5 to 2M urea, in the presence of a polypeptide and / or polynucleotide that increases the solubility, etc. The target domain includes an endosomal dissolution domain, such as an influenza HA domain; and other polypeptides that contribute to production, such as an IF2 domain, a GST domain, a GRPE domain, etc. The polypeptide may be formulated for improved stability. For example, the peptides may be PEGylated, wherein the polyethyleneoxy group provides increased longevity in the blood stream.

[0232] Additionally or alternatively, the CasZ polypeptide of the present disclosure may be fused to a polypeptide penetration domain to facilitate absorption by cells. Many penetration domains are known in the art and can be used for non-integrated polypeptides of the present disclosure, including peptides, peptide mimetics, and non-peptide carriers. For example, the penetrating peptide may be derived from the third alpha helix of the Drosophila melanogaster transcription factor antennapedia gene (referred to as penetrating protein), the third alpha helix comprising the amino acid sequence RQIKIWFQNRRMKWKK (SEQ ID NO: 134). As another example, the penetrating peptide comprises an HIV-1tat basic region amino acid sequence, which may include, for example, amino acids 49-57 of naturally occurring tat proteins. Other penetration domains include polyarginine motifs, such as regions of amino acids 34-56 of HIV-1rev proteins, nine arginines, eight arginines, 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. USA 2000 Nov 21; 97(24):13003-8; published U.S. patent applications 20030220334; 20030083256; 20030032593; and 20030022831, expressly incorporated herein by reference for their teachings of translocation peptides and peptoids). The nona-arginine (R9) sequence is one of the more effective PTDs that has been characterized (Wender et al. 2000; Uemura et al. 2002). The site for fusion may be selected so as to optimize the biological activity, secretion or binding characteristics of the polypeptide. The optimal site will be determined by routine experimentation.

[0233] The CasZ polypeptide of the present disclosure can be produced in vitro or by eukaryotic cells or by prokaryotic cells, and it can be further processed by unfolding (e.g., heat denaturation, dithiothreitol reduction, etc.), and can be further refolded using methods known in the art.

[0234] Targeted modifications that do not alter the primary sequence include chemical derivatization of the polypeptide, such as acylation, acetylation, carboxylation, amidation, etc. Also included are modifications of glycosylation, such as those performed by modifying the glycosylation form of the polypeptide during the synthesis and processing of the polypeptide or in a further processing step; for example, those performed by exposing the polypeptide to an enzyme that affects glycosylation (such as a mammalian glycosylase or deglycosylase). Also encompassed are sequences with phosphorylated amino acid residues such as phosphotyrosine, phosphoserine, or phosphothreonine.

[0235] Also suitable for inclusion in the embodiments of the present disclosure are nucleic acids (e.g., nucleic acids encoding CasZ guide RNAs, nucleic acids encoding CasZ fusion proteins, etc.) and proteins (e.g., CasZ fusion proteins derived from wild-type proteins or variant proteins) that have been modified using common molecular biology techniques and synthetic chemistry to improve their resistance to proteolytic degradation, change target sequence specificity, optimize solubility characteristics, change protein activity (e.g., transcriptional regulatory activity, enzymatic activity, etc.) or make them more suitable. Analogs of such polypeptides include those containing residues other than naturally occurring L-amino acids (e.g., D-amino acids or non-naturally occurring synthetic amino acids). D-amino acids may replace some or all of the amino acid residues.

[0236] The CasZ polypeptides of the present disclosure can be prepared by in vitro synthesis using conventional methods as known in the art. Various commercial synthesis devices can be used, such as automatic synthesizers from Applied Biosystems, Inc., Beckman, etc. By using a synthesizer, naturally occurring amino acids can be replaced by non-natural amino acids. The specific order and manner of preparation will be determined by convenience, economy, required purity, etc.

[0237] If desired, various groups can be introduced into the peptide during synthesis or during expression, which allow attachment to other molecules or surfaces. Thus, cysteine ​​can be used to prepare thioethers, histidine for attachment to metal ion complexes, carboxyl groups for the formation of amides or esters, amino groups for the formation of amides, etc.

[0238] The CasZ polypeptides of the present disclosure can also be separated and purified according to conventional recombinant synthesis methods. Lysates can be prepared by expression hosts, and lysates are purified using high performance liquid chromatography (HPLC), exclusion chromatography, gel electrophoresis, affinity chromatography or other purification techniques. In most cases, the composition used will account for 20% by weight or more of the desired product, more generally 75% by weight or more, preferably 95% by weight or more, and for therapeutic purposes, it is usually 99.5% by weight or more, relative to pollutants associated with the method for preparing the product and purifying it. Typically, the percentage will be based on total protein. Therefore, in some cases, the CasZ polypeptides or CasZ fusion polypeptides of the present disclosure have at least 80% purity, at least 85% purity, at least 90% purity, at least 95% purity, at least 98% purity or at least 99% purity (e.g., free of pollutants, non-CasZ proteins or other macromolecules, etc.).

[0239] 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, the cells can be provided with CasZ guide RNA and / or CasZ polypeptide and / or CasZtrancRNA and / or donor template sequence (whether they are introduced as a nucleic acid or a polypeptide) for about 30 minutes to about 24 hours, such as 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 time period from about 30 minutes to about 24 hours, which can be repeated at a frequency of about every day to about every 4 days, such as every 1.5 days, every 2 days, every 3 days, or any other frequency from about every day to about every four days. One or more agents can be provided to the subject cells one or more times (e.g., once, twice, three times, or more than three times), and after each contact event the cells are allowed to incubate with the one or more agents for a certain amount of time, e.g., 16-24 hours, after which time the culture medium is replaced with fresh culture medium and the cells are further cultured.

[0240] In the case where two or more different targeting complexes (e.g., two different CasZ guide RNAs complementary to different sequences within the same or different target nucleic acids) are provided to the cell, the complexes may be provided simultaneously (e.g., as two polypeptides and / or nucleic acids) or delivered simultaneously. Alternatively, the complexes may be provided sequentially, e.g., first providing a targeting complex, then providing a second targeting complex, etc., or vice versa.

[0241] In order to improve the delivery of DNA vectors to target cells, DNA can be protected from damage and DNA can be promoted to enter cells, for example, by using lipoplexes and polyplexes. Therefore, in some cases, the nucleic acids of the present disclosure (e.g., recombinant expression vectors of the present disclosure) can be covered with lipids in an organized structure (like micelles or liposomes). When an 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 using cationic lipids have been shown to be useful for gene transfer. Cationic lipids naturally complex with negatively charged DNA due to their positive charge. Also due to their charge, they interact with cell membranes. Then endocytosis of the lipoplex occurs, and DNA is released into the cytoplasm. Cationic lipids can also prevent cells from degrading DNA.

[0242] Complexes of polymers with DNA are called polyplexes. Most polyplexes consist of cationic polymers, and their production is regulated by ionic interactions. A huge difference between the methods of action of polyplexes and lipoplexes is that polyplexes cannot release their DNA cargo into the cytoplasm, for which co-transfection with an endosomolytic agent (which dissolves endosomes generated during endocytosis) such as inactivated adenovirus must occur. However, this is not always the case; polymers such as polyethyleneimine have their own methods of endosomal disruption, as do chitosan and trimethyl chitosan.

[0243] Dendrimers, a spherical, highly branched macromolecule, can also be used to genetically modify stem cells. The surface of the dendrimer particles can be functionalized to change their properties. In particular, it is possible to construct cationic dendrimers (i.e., dendrimers with a positive surface charge). When genetic material (such as a DNA plasmid) is present, charge complementarity leads to a temporary association of nucleic acids with cationic dendrimers. Upon reaching their destination, the dendrimer-nucleic acid complex can be taken up into the cell by endocytosis.

[0244] In some cases, the nucleic acid (e.g., expression vector) of the present disclosure comprises an insertion site for a target guide sequence. For example, a nucleic acid may comprise an insertion site for a target guide sequence, wherein the insertion site is adjacent to a nucleotide sequence encoding a portion of a CasZ guide RNA, and when the guide sequence is altered and hybridized with a desired target sequence (e.g., a sequence that contributes to the CasZ binding aspects of the guide RNA, e.g., a sequence that contributes to one or more dsRNA duplexes of the CasZ guide RNA-this portion of the guide RNA may also be referred to as the 'scaffold' or 'constant region' of the guide RNA), the portion of the CasZ guide RNA will not change. Therefore, in some cases, a subject nucleic acid (e.g., expression vector) comprises a nucleotide sequence encoding a CasZ guide RNA, except that the portion of the guide sequence portion encoding the guide RNA is an insertion sequence (insertion site). An insertion site is any nucleotide sequence for inserting a desired sequence. An "insertion site" for various techniques is known to those of ordinary skill in the art, and any convenient insertion site may be used. An insertion site may be used for any method of manipulating a nucleic acid sequence. For example, in some cases, the insertion site is a multiple cloning site (MCS) (e.g., a site comprising one or more restriction enzyme recognition sequences), a site for ligation-independent cloning, a site for recombination-based cloning (e.g., att site-based recombination), a nucleotide sequence recognized by CRISPR / Cas (e.g., Cas9)-based technology, etc.

[0245] 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 restriction enzyme recognition sequences are contained), whether the site includes a target site for a CRISPR / Cas protein, etc.). In some cases, the insertion site of the 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 length of the insertion site of the subject nucleic acid has a length in the range of 2 to 50 nucleotides (nt) (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 some cases, the length of the insertion site of the subject nucleic acid has a length in the range of 5 to 40 nt.

[0246] Nucleic acid modification

[0247] In some embodiments, the subject nucleic acid (e.g., CasZ guide RNA or trancRNA) has one or more modifications (e.g., base modification, backbone modification, etc.) to provide new or enhanced features (e.g., improved stability) to the nucleic acid. Nucleoside is a base-sugar combination. The base portion of a nucleoside is generally a heterocyclic base. The two most common categories of such heterocyclic bases are purine and pyrimidine. Nucleotide is a nucleoside that also comprises a phosphate group covalently attached to the sugar portion of a nucleoside. For those nucleosides comprising pentofuranosyl, the phosphate group may be attached to the 2', 3' or 5' hydroxyl portion of the sugar. In forming an oligonucleotide, a phosphate group covalently attaches nucleosides adjacent to each other to form a linear polymer compound. Then, each end of this linear polymer compound may be further connected to form a cyclic compound, however, a linear compound is suitable. In addition, a linear compound may have internal nucleotide base complementarity and may therefore be folded in order to produce a completely or partially double-stranded compound. In an oligonucleotide, a phosphate group is generally referred to as forming an internucleoside backbone of an oligonucleotide. The normal bond or backbone of RNA and DNA is a 3' to 5' phosphodiester bond.

[0248] Suitable nucleic acid modifications include, but are not limited to, 2'O-methyl modified nucleotides, 2'fluorine modified nucleotides, locked nucleic acid (LNA) modified nucleotides, peptide nucleic acid (PNA) modified nucleotides, nucleotides with phosphorothioate bonds, and 5' caps (e.g., 7-methylguanylate caps (m7G)). Additional details and additional modifications are described below.

[0249] 2'-O-methyl modified nucleotides (also known as 2'-O-methyl RNA) are naturally occurring RNA modifications found in tRNA and other small RNAs that occur as post-transcriptional modifications. Oligonucleotides containing 2'-O-methyl RNA can be directly synthesized. This modification increases the Tm of the RNA: RNA duplex, but only results in slight changes in RNA: DNA stability. It is stable to attack by single-stranded ribonucleases, and its susceptibility to DNA enzymes is typically 5 to 10 times lower than that of DNA. It is commonly used in antisense oligonucleotides as a means of increasing stability and binding affinity for target messengers.

[0250] 2' fluorine-modified nucleotides (e.g., 2' fluorine bases) have fluorine-modified ribose sugars that increase binding affinity (Tm) and also confer a degree of relative nuclease resistance compared to native RNA. These modifications are often used in ribozymes and siRNAs to improve stability in serum or other biological fluids.

[0251] LNA bases have a modification to the ribose backbone that locks the base in a C3'-internal position, which favors RNA A-type helical duplex geometry. This modification significantly increases Tm and also has very strong nuclease resistance. Multiple LNA insertions can be placed in any position in an oligonucleotide except the 3' end. Applications from antisense oligonucleotides to hybridization probes to SNP detection and allele-specific PCR have been described. Due to the large increase in Tm conferred by LNA, they can also cause an increase in primer dimer formation and the formation of self-hairpins. In some cases, the number of LNAs incorporated into a single oligonucleotide is 10 bases or less.

[0252] Phosphorothioate (PS) bonds (i.e., phosphorothioate linkages) replace non-bridging oxygens in the phosphate backbone of a nucleic acid (e.g., an oligonucleotide) with a sulfur atom. This modification renders the internucleotide bond resistant to nuclease degradation. Phosphorothioate bonds can be introduced between the last 3-5 nucleotides at the 5' or 3' end of an oligonucleotide to inhibit exonuclease degradation. Inclusion of phosphorothioate bonds within an oligonucleotide (e.g., throughout an oligonucleotide) can also help reduce attack by endonucleases.

[0253] In some embodiments, the subject nucleic acid has one or more nucleotides, and the one or more nucleotides are 2'-O-methyl modified nucleotides. In some embodiments, the subject nucleic acid (e.g., guide RNA, tranc RNA, etc.) has one or more 2' fluorine modified nucleotides. In some embodiments, the subject nucleic acid (e.g., dsRNA, siNA, etc.) has one or more LNA bases. In some embodiments, the subject nucleic acid (e.g., dsRNA, siNA, etc.) has one or more nucleotides connected by thiophosphate bonds (i.e., the subject nucleic acid has one or more thiophosphate bonds). In some embodiments, the subject nucleic acid (e.g., dsRNA, siNA, etc.) has a 5' cap (e.g., 7-methylguanylate cap (m7G)). In some embodiments, the subject nucleic acid (e.g., guide RNA, tranc RNA, etc.) has a combination of modified nucleotides. For example, a subject nucleic acid (e.g., a guide RNA, a tranc RNA, etc.) can have a 5' cap (e.g., a 7-methylguanylate cap (m7G)) in addition to having one or more nucleotides having other modifications (e.g., 2'-O-methyl nucleotides and / or 2' fluoro-modified nucleotides and / or LNA bases and / or phosphorothioate linkages).

[0254] Modified Backbones and Modified Internucleoside Linkages

[0255] Examples of suitable nucleic acids (e.g., CasZ guide RNA and / or CasZ trancRNA) containing modifications include nucleic acids containing modified backbones or non-natural internucleoside linkages. Nucleic acids with modified backbones include those nucleic acids that retain phosphorus atoms in the backbone and those nucleic acids that do not have phosphorus atoms in the backbone.

[0256] The oligonucleotide backbone of suitable modification containing phosphorus atom comprises for example, phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, methyl and other alkyl phosphates (comprising 3'-alkylene phosphate, 5'-alkylene phosphate and chiral phosphate), phosphinate, phosphoramidate (comprising 3'-aminophosphoramidate and aminoalkylphosphoramidate), diaminophosphoroate, thionylphosphoramidate, thionylphosphoratotriester, selenophosphoroester and boronophosphoroester with normal 3'-5' linkage, 2'-5' connection analogs of these materials and those oligonucleotide backbones with reverse polarity, wherein one or more internucleotide linkages are 3' to 3', 5' to 5' or 2' to 2' linkages. Suitable oligonucleotide with reverse polarity comprises single 3' to 3' linkage at the most 3' internucleotide linkage, i.e., single reverse nucleoside residue that can be basic (nucleobase is lost or it is replaced by hydroxyl). Also included are various salts (such as, for example, potassium or sodium), mixed salts and free acid forms.

[0257] In some embodiments, the subject nucleic acids contain one or more phosphorothioate and / or heteroatom internucleoside linkages, specifically -CH 2 -NH-O-CH 2 -、-CH 2 -N(CH 3 )-O-CH 2 - (called methylene (methylimino) or MMI skeleton), -CH 2 -ON(CH 3 )-CH 2 -、 -CH 2 -N(CH 3 )-N(CH 3 )-CH 2 - and -ON(CH 3 )-CH 2 -CH 2 -(wherein the natural phosphodiester internucleotide linkage is represented as -OP(=O)(OH)-O-CH 2 -). MMI-type internucleoside linkages are disclosed in the above-mentioned U.S. Patent No. 5,489,677, the disclosure of which is incorporated herein by reference in its entirety. Suitable amide internucleoside linkages are disclosed in U.S. Patent No. 5,602,240, the disclosure of which is incorporated herein by reference in its entirety.

[0258] Also suitable is the nucleic acid with morpholino backbone structure, as described in for example U.S. Patent number 5,034,506.For example, in some embodiments, theme nucleic acid comprises 6 yuan of morpholino rings that replace ribose ring.In some embodiments of these embodiments, diaminophosphoroester or other non-phosphodiester internucleoside linkage replaces phosphodiester linkage.

[0259] Suitable modified polynucleotide backbones that do not contain phosphorus atoms have backbones formed by short-chain alkyl or cycloalkyl nucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl nucleoside linkages, or one or more short-chain heteroatoms or heterocyclic nucleoside linkages. These include: those backbones with morpholino linkages (partially formed by the sugar portion of the nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formyl acetyl and thioformyl acetyl backbones; methylene formyl acetyl and thioformyl acetyl backbones; riboacetyl backbones; olefin-containing backbones; sulfamic acid ester backbones; methylene imino and methylene hydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and those with mixed N, O, S and CH 2 Other skeletons of components.

[0260] Simulants

[0261] The subject nucleic acid can be a nucleic acid mimic.When the term "simulations" is applied to polynucleotides, it is intended to include polynucleotides in which only furanose rings or both furanose rings and internucleotide linkages are replaced by non-furanose groups, and only furanose ring replacement is also referred to as sugar replacement in the art.Maintain heterocyclic base moiety or modified heterocyclic base moiety for hybridization with appropriate target nucleic acid.A kind of such nucleic acid (polynucleotide mimics having shown excellent hybridization properties) is called peptide nucleic acid (PNA).In PNA, the sugar backbone of polynucleotides is replaced by an amide-containing backbone, specifically by an aminoethylglycine backbone.Nucleotide is retained and directly or indirectly bonded to the aza nitrogen atom of the amide moiety of the backbone.

[0262] One polynucleotide mimetic that has been reported to have excellent hybridization properties is peptide nucleic acid (PNA). The backbone in the PNA compound is two or more linked aminoethylglycine units that give the PNA an amide-containing backbone. The heterocyclic base moiety is directly or indirectly bonded to the aza nitrogen atom of the amide portion of the backbone. Representative U.S. patents describing 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 herein by reference in their entirety.

[0263] Another class of polynucleotide mimics studied is based on connecting morpholino units (morpholino nucleic acids) with heterocyclic bases connected to the morpholino ring. Many linking groups for connecting morpholino monomeric units in morpholino nucleic acids have been reported. A class of linking groups has been selected to obtain nonionic oligomeric compounds. Oligomeric compounds based on nonionic morpholinos are unlikely to have undesirable interactions with cell proteins. Morpholino-based polynucleotides are nonionic mimics of oligonucleotides that are unlikely to form undesirable interactions with cell 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 herein by reference in its entirety. A variety of compounds in morpholino-based polynucleotides have been prepared, with a variety of different linking groups connecting monomer subunits.

[0264] Another class of polynucleotide mimics is called cyclohexenyl nucleic acid (CeNA). The furanose ring usually present in DNA / RNA molecules is replaced by a cyclohexenyl ring. CeNADMT protected phosphoramidite monomers have been prepared and used for the synthesis of oligomeric compounds according to the classical phosphoramidite chemistry. Fully modified CeNA oligomeric compounds and oligonucleotides with specific positions modified with CeNA have been prepared and studied (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 speaking, the incorporation of CeNA monomers into DNA chains increases the stability of DNA / RNA hybrids. CeNA oligoadenylates form complexes with RNA and DNA complementary sequences that have stability similar to that of natural complexes. Studies showing the incorporation of CeNA structures into natural nucleic acid structures by NMR and circular dichroism continue with simple conformational adjustments.

[0265] Another modification includes locked nucleic acids (LNA), in which the 2'-hydroxyl group is linked to the 4' carbon atom of the sugar ring to form a 2'-C, 4'-C-oxymethylene linkage, thereby forming a bicyclic sugar moiety. The linkage can be a methylene (-CH 2 -), i.e., a group bridging the 2' oxygen atom and the 4' carbon atom, wherein 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). LNA and LNA analogs exhibit very high duplex thermal stability (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 LNA have been described (e.g., Wahlestedt et al., Proc. Natl. Acad. Sci. USA, 2000, 97, 5633-5638, the disclosure of which is incorporated herein by reference in its entirety).

[0266] The synthesis and preparation of LNA monomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine and uracil, as well as 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). LNA and its preparation are also described in WO98 / 39352 and WO99 / 14226 and U.S. applications 20120165514, 20100216983, 20090041809, 20060117410, 20040014959, 20020094555 and 20020086998, the disclosures of which are incorporated herein by reference in their entirety.

[0267] Modified sugar moiety

[0268] The subject nucleic acid may also contain one or more substituted sugar moieties. Suitable polynucleotides contain a sugar substituent group selected from the group consisting of: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, wherein alkyl, alkenyl, and alkynyl groups may be substituted or unsubstituted C 1 To C 10 Alkyl or C 2 To C 10 Particularly suitable are: O((CH 2 ) n O) m CH 3 、O(CH 2 ) n OCH 3 , O(CH 2 ) n NH 2 , O(CH 2 ) n CH 3 、 O(CH 2 ) n ONH 2 and O(CH 2 ) n ON((CH 2 ) n CH 3 ) 2 , wherein n and m are from 1 to about 10. Other suitable polynucleotides contain a sugar substituent selected from the group consisting of: C 1 To C 10 Lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 、OCN、Cl、Br、CN、CF 3 、OCF 3 、SOCH 3 、SO 2 CH 3 、ONO 2 、NO 2 、N 3 NH 2 , heterocycloalkyl, heterocycloalkylaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage group, reporter group, intercalator, group that improves the pharmacokinetic properties of oligonucleotides, or group that improves the pharmacodynamic properties of oligonucleotides, and other substituents with similar properties. Suitable modifications include 2'-methoxyethoxy (2'-O-CH2 CH 2 OCH 3 , 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'-dimethylaminooxyethoxy, i.e., O(CH 2 ) 2 ON(CH 3 ) 2 The 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-CH 2 -O-CH 2 -N(CH 3 ) 2 .

[0269] Other suitable sugar substituents include methoxy (—O—CH 3 ), aminopropyloxy (--OCH 2 CH 2 CH 2 NH 2 ), allyl (-CH 2 -CH=CH 2 ), -O-allyl (--O--CH 2 —CH=CH 2 ) and fluorine (F). The 2'-sugar substitution group may be in the arabinose (upper) position or the ribose (lower) position. A suitable 2'-arabinose modification is 2'-F. Similar modifications may also be made at other positions on the oligomeric compound, specifically at the 3' terminal nucleoside of the sugar or at the 3' position in a 2'-5' linked oligonucleotide and at the 5' position of the 5' terminal nucleotide. The oligomeric compound may also have a sugar mimetic such as a cyclobutyl moiety in place of the pentofuranosyl sugar.

[0270] Base modification and substitution

[0271] The subject nucleic acids may also include modifications or substitutions of nucleobases (often referred to in the art as "bases"). 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 derivatives of adenine and guanine and other alkyl derivatives, 2-propyl derivatives of adenine and guanine and other alkyl derivatives, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C=C-CH 3 ) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-sulfanyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halo (particularly 5-bromo), 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 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-clamps such as substituted phenoxazine cytidines (e.g., 9-(2-aminoethoxy)-H-pyrimido(5,4-(b)(1,4)benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido(4,5-b)indol-2-one), pyridoindole cytidine (H-pyrido(3',2':4,5)pyrrolo(2,3-d)pyrimidin-2-one).

[0272] Heterocyclic base moieties may also include those bases in which purine or pyrimidine bases are replaced by other heterocycles, such as 7-deazaadenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Other nucleobases include those disclosed in U.S. Patent No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, 858-859 pages, Kroschwitz, JI compiles John Wiley & Sons, 1990, by Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613 disclosed those and by Sanghvi, YS, Chapter 15, Antisense Research and Applications, 289-302 pages, Crooke, ST and Lebleu, B. compile, CRC Press, 1993 disclosed those, the disclosures of these documents are incorporated herein by reference in their entirety. Some of these nucleobases can be used to increase the binding affinity of 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°C-1.2°C (Sanghvi et al., ed. Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278; the disclosure of which is incorporated herein by reference in its entirety) and are suitable base substitutions, for example, when combined with 2'-O-methoxyethyl sugar modifications.

[0273] Conjugate

[0274] Another possible modification of the subject nucleic acid involves one or more parts or conjugates that enhance the activity, cell distribution or cell absorption of the oligonucleotides to be chemically connected to the polynucleotides. These parts or conjugates may include conjugate groups covalently bonded to functional groups such as primary hydroxyl or secondary hydroxyl. 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 pharmacodynamic properties of oligomers. Suitable conjugate groups include but are not limited to cholesterol, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin and dyes. The group that enhances the pharmacodynamic properties includes improving absorption, enhancing resistance to degradation and / or strengthening the group of sequence-specific hybridization with the target nucleic acid. The group that enhances the pharmacodynamic properties includes improving the absorption, distribution, metabolism or excretion of the subject nucleic acid.

[0275] Conjugate moieties include, but are not limited to, lipid moieties such as cholesterol moieties (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), thioethers such as hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), aliphatic chains such as dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), phospholipids such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), polyamines or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-hydroxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937).

[0276] The conjugate may include a "protein transduction domain" or PTD (also known as CPP-cell penetrating peptide), which may refer to a polypeptide, polynucleotide, carbohydrate, or organic or inorganic compound that facilitates traversal of a lipid bilayer, micelle, cell membrane, organelle membrane, or vesicle membrane. The PTD attached to another molecule (which may range from a small polar molecule to a large macromolecule and / or nanoparticle) facilitates the molecule to traverse the membrane, for example, from the extracellular space into the intracellular space or from the cytosol into an organelle (e.g., a nucleus). In some embodiments, the PTD is covalently linked to the 3' end of the exogenous polynucleotide. In some embodiments, the PTD is covalently linked to the 5' end of the exogenous polynucleotide. Exemplary PTDs include, but are not limited to, a minimal eleven amino acid polypeptide protein transduction domain (corresponding to residues 47-57 of HIV-1 TAT comprising YGRKKRRQRRR; SEQ ID NO: 130); a polyarginine sequence comprising a sufficient number of arginines (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10-50 arginines) to directly enter the cell; a VP22 domain (Zender et al. (2002) Cancer Cell Biology, 1999; 9(6):489-96); Drosophila Antennapedia gene protein transduction domain (Noguchi et al. (2003) Diabetes 52(7):1732-1737); truncated human calcitonin peptide (Trehin et al. (2004) Pharm. Research 21:1248-1256); polylysine (Wender et al. (2000) Proc. Natl. Acad. Sci. USA 97:13003-13008); RRQRRTSKLMKR SEQ ID NO:131); transport protein GWTLNSAGYLLGKINLKALAALAKKIL SEQ ID NO:132); KALAWEAKLAKALAKALAKHLAKALAKALKCEA SEQ ID NO:133); and RQIKIWFQNRRMKWKK SEQ ID NO:134). Exemplary PTDs include, but are not limited to: YGRKKRRQRRRSEQ ID NO: 130); RKKRRQRRRSEQ ID NO: 135); arginine homopolymers having 3 to 50 arginine residues; exemplary PTD domain amino acid sequences include, but are not limited to, any one of the following sequences: YGRKKRRQRRRSEQ ID NO: 130); RKKRRQRRSEQ ID NO: 136); YARAAARQARASEQ ID NO: 137); THRLPRRRRRRSEQ ID NO: 138); and GGRRARRRRRRSEQ ID NO: 139).In some cases, the PTD is an activatable CPP (ACPP) (Aguilera et al. (2009) Integr Biol (Camb) June; 1(5-6): 371-381). ACPPs include a polycationic CPP (e.g., Arg9 or "R9") linked to a matching polyanion (e.g., Glu9 or "E9") via a cleavable linker, 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 polyarginine and its inherent adhesive properties, thereby "activating" the ACPP to traverse the membrane.

[0277] Introduction of components into target cells

[0278] CasZ guide RNA (or a nucleic acid comprising a nucleotide sequence encoding a CasZ guide RNA) and / or CasZ polypeptide (or a nucleic acid comprising a nucleotide sequence encoding a CasZ polypeptide) and / or CasZ trancRNA (or a nucleic acid comprising a nucleotide sequence encoding a CasZ trancRNA) and / or a donor polynucleotide (donor template) can be introduced into a host cell by any of a variety of well-known methods.

[0279] Any of a variety of compounds and methods can be used to deliver the CasZ system of the present disclosure to target cells. As a non-limiting example, the CasZ system of the present disclosure can be combined with lipids. As another non-limiting example, the CasZ system of the present disclosure can be combined with particles or formulated into particles.

[0280] Methods for 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., an expression construct / vector) into a target cell (e.g., a prokaryotic cell, a eukaryotic cell, a plant cell, an animal cell, a mammalian cell, a human cell, etc.). Suitable methods include, for example, viral infection, transfection, conjugation, protoplast fusion, liposome transfection, electroporation, calcium phosphate precipitation, polyethyleneimine (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), etc.

[0281] In some cases, the CasZ polypeptides of the present disclosure (e.g., wild-type proteins, variant proteins, chimeric / fusion proteins, dCasZ, etc.) are provided as nucleic acids encoding CasZ polypeptides (e.g., mRNA, DNA, plasmids, expression vectors, viral vectors, etc.). In some cases, the CasZ polypeptides of the present disclosure are provided directly as proteins (e.g., not with associated guide RNAs or with associated guide RNAs, i.e., as ribonucleoprotein complexes). The CasZ polypeptides of the present disclosure may be introduced into cells (provided to cells) by any convenient method; such methods are known to those of ordinary skill in the art. As an illustrative example, the CasZ fusion polypeptides of the present disclosure may be injected directly into cells (e.g., with or without CasZ guide RNAs or nucleic acids encoding CasZ guide RNAs, and with or without donor polynucleotides, and with or without CasZ trancRNA). As another example, a preformed complex (RNP) of a CasZ polypeptide of the present disclosure and a CasZ guide RNA can be introduced into a cell (e.g., a eukaryotic cell) (e.g., by injection, by nuclear transfection; by a protein transduction domain (PTD) conjugated to one or more components, e.g., conjugated to a CasZ protein, conjugated to a guide RNA, conjugated to a CasZtrancRNA, conjugated to a CasZ polypeptide of the present disclosure and a guide RNA; etc.).

[0282] In some cases, nucleic acids (e.g., CasZ guide RNA and / or nucleic acids encoding CasZ guide RNA, nucleic acids encoding CasZ proteins, CasZ trancRNA and / or nucleic acids encoding CasZ trancRNA, etc.) and / or polypeptides (e.g., CasZ polypeptides; CasZ fusion polypeptides) are delivered to cells (e.g., target host cells) in or associated with particles. In some cases, the CasZ system of the present disclosure is delivered to cells in or associated with particles. The terms "particle" and "nanoparticle" may be used interchangeably as appropriate. For example, a recombinant expression vector comprising a nucleotide sequence encoding a CasZ polypeptide of the present disclosure and / or a CasZ guide RNA, an mRNA comprising a nucleotide sequence encoding a CasZ polypeptide of the present disclosure, and a guide RNA can be delivered simultaneously using particles or lipid envelopes; for example, a CasZ polypeptide and a CasZ guide RNA and / or a trancRNA, for example as a complex (e.g., a ribonucleoprotein (RNP) complex) can be delivered by particles, for example, by delivery particles comprising a lipid or a lipidoid and a hydrophilic polymer (e.g., a cationic lipid and a hydrophilic polymer), for example, wherein the cationic lipid comprises 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or 1,2-dioctanoyl-sn-glycero-3-phosphocholine (DMPC) and / or wherein the hydrophilic polymer comprises ethylene glycol or polyethylene glycol (PEG); and / or wherein the particle further comprises cholesterol (e.g., particles from formulation 1 = DOTAP 100, DMPC 0, PEG 0, cholesterol 0; formulation number 2 = =DOTAP90, DMPC0, PEG10, cholesterol 0; Formulation No. 3 =DOTAP90, DMPC0, PEG5, cholesterol 5). For example, a multi-step method can be used to form particles, wherein the CasZ polypeptide and the CasZ guide RNA are mixed together, for example, at a 1:1 molar ratio, for example, at room temperature, for example, for 30 minutes, for example, in sterile nuclease-free 1× phosphate buffered saline (PBS); and DOTAP, DMPC, PEG, and cholesterol suitable for the formulation are separately dissolved in alcohol (e.g., 100% ethanol), and the two solutions are mixed together to form particles containing the complex).

[0283] The CasZ polypeptides of the present disclosure (or mRNAs comprising nucleotide sequences encoding the CasZ polypeptides of the present disclosure; or recombinant expression vectors comprising nucleotide sequences encoding the CasZ polypeptides of the present disclosure) and / or CasZ guide RNAs (or nucleic acids, such as one or more expression vectors encoding CasZ guide RNAs) can be delivered simultaneously using particles or lipid envelopes. For example, nanoparticles having a biodegradable core-shell structure of a poly(β-amino ester) (PBAE) core encapsulated by a phospholipid bilayer shell can be used. In some cases, particles / nanoparticles based on self-assembling bioadhesive polymers are used; such particles / nanoparticles can be applied to oral delivery of peptides, intravenous delivery of peptides, and intranasal delivery of peptides, such as delivery to the brain. Other embodiments are also contemplated, such as oral absorption and ocular delivery of hydrophobic drugs. Molecular envelope technology can be used, which involves an engineered polymer envelope that is protected and delivered to the site of disease. Doses of about 5 mg / kg can be used in single or multiple doses, depending on various factors, such as the target tissue.

[0284] Lipidoid compounds (e.g., as described in U.S. Patent Application 20110293703) can also be used for the administration of polynucleotides, and can be used to deliver CasZ polypeptides of the present disclosure, CasZ fusion polypeptides of the present disclosure, RNPs of the present disclosure, nucleic acids of the present disclosure, or CasZ systems of the present disclosure. On the one hand, amino alcohol lipidoid compounds are combined with agents to be delivered to cells or subjects to form microparticles, nanoparticles, liposomes, or micelles. Amino alcohol lipidoid compounds can be combined with other amino alcohol lipidoid compounds, polymers (synthetic or natural), surfactants, cholesterol, carbohydrates, proteins, lipids, etc. to form particles. These particles can then be optionally combined with pharmaceutical excipients to form a pharmaceutical composition.

[0285] Poly (β-amino alcohol) (PBAA) can be used to deliver the CasZ polypeptide of the present disclosure, the CasZ fusion polypeptide of the present disclosure, the RNP of the present disclosure, the nucleic acid of the present disclosure (e.g., CasZ guide RNA and / or CasZ trancRNA) or the CasZ system of the present disclosure to a target cell. U.S. Patent Publication No. 20130302401 relates to a class of poly (β-amino alcohol) (PBAA) prepared using combinatorial polymerization.

[0286] Sugar-based particles, for example, GalNAc as described in WO2014118272 (incorporated herein by reference) and Nair, JK et al., 2014, Journal of the American Chemical Society 136(49), 16958-16961) can be used to deliver the CasZ polypeptide of the present disclosure, the CasZ fusion polypeptide of the present disclosure, the RNP of the present disclosure, the nucleic acid of the present disclosure or the CasZ system of the present disclosure to target cells.

[0287] In some cases, lipid nanoparticles (LNPs) are used to deliver CasZ polypeptides of the present disclosure, CasZ fusion polypeptides of the present disclosure, RNPs of the present disclosure, nucleic acids of the present disclosure (e.g., CasZ guide RNA and / or CasZ trancRNA) or CasZ systems of the present disclosure to target cells. Negatively charged polymers (such as RNA) can be loaded into LNPs at low pH values ​​(e.g., pH 4), where ionizable lipids display positive charges. However, at physiological pH values, LNPs exhibit low surface charges compatible with longer circulation times. Four ionizable cationic lipids have been considered, namely 1,2-dilinoleoyl-3-dimethylammonium-propane (DLinDAP), 1,2-dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxy-keto-N,N-dimethyl-3-aminopropane (DLinKDMA) and 1,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA). The preparation of LNPs is described, for example, in Rosin et al. (2011) Molecular Therapy 19: 1286-2200). Cationic lipids 1,2-dilinoleoyl-3-dimethylammonium-propane (DLinDAP), 1,2-dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxyketo-N,N-dimethyl-3-aminopropane (DLinK-DMA), 1,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA), (3-o-[2″-(methoxypolyethylene glycol 2000) succinyl]-1,2-dimyristoyl-sn-ethylene glycol (PEG-S-DMG), and R-3-[(.ω.- Methoxy-poly (ethylene glycol) 2000) carbamoyl] -1,2- dimyristoyloxypropyl -3-amine (PEG-C-DOMG). Nucleic acids (e.g., CasZ guide RNA; nucleic acids disclosed herein, etc.) can be encapsulated in LNPs containing DLinDAP, DLinDMA, DLinK-DMA, and DLinKC2-DMA (cationic lipid: DSPC: CHOL: PEGS-DMG or PEG-C-DOMG molar ratio of 40:10:40:10). In some cases, 0.2% SP-DiOC18 is incorporated.

[0288] Spherical nucleic acid (SNA) TM) constructs and other nanoparticles (particularly gold nanoparticles) can be used to deliver the CasZ polypeptides of the present disclosure, the CasZ fusion polypeptides of the present disclosure, the RNPs of the present disclosure, the nucleic acids of the present disclosure (e.g., CasZ guide RNA and / or CasZ trancRNA) or the CasZ system of the present disclosure to target cells. See, e.g., Cutler et al., J. Am. Chem. Soc. 2011 133:9254-9257; Hao et al., Small. 2011 7:3158-3162; Zhang et al., ACS Nano. 2011 5:6962-6970; Cutler et al., J. Am. Chem. Soc. 2012 134:1376-1391; Young et al., Nano Lett. 2012 12:3867-71; Zheng et al., Proc. Natl. Acad. Sci. USA. 2012 109:11975-80; Mirkin, Nanomedicine 2012 7:635-638; Zhang et al., J. Am. Chem. Soc. 2012 134:16488-1691; Weintraub, Nature 2013495:S14-S16; Choi et al., Proc. Natl. Acad. Sci. USA. 2013110(19):7625-7630; Jensen et al., Sci. Transl. Med. 5, 209ra152 (2013) and Mirkin, et al., Small, 10:186-192.

[0289] Self-assembled nanoparticles with RNA can be constructed with polyethyleneimine (PEI) PEGylated with an Arg-Gly-Asp (RGD) peptide ligand attached at the distal end of polyethylene glycol (PEG).

[0290] Generally speaking, "nanoparticle" refers to any particle with a diameter less than 1000nm. In some cases, the nanoparticles suitable for delivering the CasZ polypeptide of the present disclosure, the CasZ fusion polypeptide of the present disclosure, the RNP of the present disclosure, the nucleic acid of the present disclosure (e.g., CasZ guide RNA and / or CasZ trancRNA) or the CasZ system of the present disclosure to the target cell have 500nm or less, for example, 25nm to 35nm, 35nm to 50nm, 50nm to 75nm, 75nm to 100nm, 100nm to 150nm, 150nm to 200nm, 200 nm to 300nm, 300nm to 400nm or 400nm to 500nm diameter. In some cases, the nanoparticles suitable for delivering the CasZ polypeptide of the present disclosure, the CasZ fusion polypeptide of the present disclosure, the RNP of the present disclosure, the nucleic acid of the present disclosure or the CasZ system of the present disclosure to the target cell have a diameter of 25nm to 200nm. In some cases, nanoparticles suitable for delivering a CasZ polypeptide of the present disclosure, a CasZ fusion polypeptide of the present disclosure, an RNP of the present disclosure, a nucleic acid of the present disclosure, or a CasZ system of the present disclosure to a target cell have a diameter of 100 nm or less. In some cases, nanoparticles suitable for delivering a CasZ polypeptide of the present disclosure, a CasZ fusion polypeptide of the present disclosure, an RNP of the present disclosure, a nucleic acid of the present disclosure, or a CasZ system of the present disclosure to a target cell have a diameter of 35 nm to 60 nm.

[0291] Nanoparticles suitable for delivering the CasZ polypeptide of the present disclosure, the CasZ fusion polypeptide of the present disclosure, the RNP of the present disclosure, the nucleic acid of the present disclosure (e.g., CasZ guide RNA and / or CasZ trancRNA) or the CasZ system of the present disclosure to target cells can be provided in different forms, for example, as solid nanoparticles (e.g., metals (such as silver, gold, iron, titanium), non-metals, lipid-based solids, polymers), suspensions of nanoparticles, or combinations thereof. Metallic, dielectric and semiconductor nanoparticles, as well as hybrid structures (e.g., core-shell nanoparticles) can be prepared. If the nanoparticles made of semiconductor materials are small enough (usually less than 10 nm) so that quantization of the electronic energy levels occurs, they can also be labeled with quantum dots. Such nanoscale particles are used as drug carriers or imaging agents in biomedical applications, and can be suitable for similar purposes in the present disclosure.

[0292] Semisolid and soft nanoparticles are also suitable for delivering CasZ polypeptides of the present disclosure, CasZ fusion polypeptides of the present disclosure, RNPs of the present disclosure, nucleic acids of the present disclosure (e.g., CasZ guide RNA and / or CasZ trancRNA) or CasZ systems of the present disclosure to target cells. A prototype nanoparticle with semisolid properties is a liposome.

[0293] In some cases, exosomes are used to deliver CasZ polypeptides of the present disclosure, CasZ fusion polypeptides of the present disclosure, RNPs of the present disclosure, nucleic acids of the present disclosure (e.g., CasZ guide RNA and / or CasZ trancRNA) or CasZ systems of the present disclosure to target cells. Exosomes are endogenous nanovesicles that transport RNA and proteins and can deliver RNA to the brain and other target organs.

[0294] In some cases, CasZ polypeptides of the present disclosure, CasZ fusion polypeptides of the present disclosure, RNPs of the present disclosure, nucleic acids of the present disclosure (e.g., CasZ guide RNA and / or CasZ trancRNA) or CasZ systems of the present disclosure are delivered to target cells using liposomes. Liposomes are spherical vesicle structures that consist of a monolayer or multilayer lipid bilayer surrounding an internal aqueous compartment and a relatively impermeable external lipophilic phospholipid bilayer. Liposomes can be made of several different types of lipids; however, phospholipids are most commonly used to generate liposomes. Although liposome formation is spontaneous when the lipid membrane is mixed with an aqueous solution, the formation of liposomes can also be accelerated by applying force in the form of shaking using a homogenizer, an ultrasonic crusher or an extrusion device. Several other additives can be added to liposomes to change their structure and properties. For example, cholesterol or sphingomyelin can be added to the liposome mixture to help stabilize the liposome structure and prevent leakage of the liposome contents (innercargo). Liposomal preparations may be composed primarily of natural phospholipids and lipids such as 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), sphingomyelin, egg phosphatidylcholine, and monosialoganglioside.

[0295] Stable nucleic acid-lipid particles (SNALP) can be used to deliver the CasZ polypeptide of the present disclosure, the CasZ fusion polypeptide of the present disclosure, the RNP of the present disclosure, the nucleic acid of the present disclosure (e.g., CasZ guide RNA and / or CasZ trancRNA) or the CasZ system of the present disclosure to target cells. The SNALP formulation may contain 2:40:10:48 molar percentages of lipid 3-N-[(methoxypoly(ethylene glycol)2000)carbamoyl]-1,2-dimyristoyloxy-propylamine (PEG-C-DMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol. SNALP liposomes can be prepared by using a lipid / siRNA ratio of 25:1 and a cholesterol / D-Lin-DMA / DSPC / PEG-C-DMA molar ratio of 48 / 40 / 10 / 2 D-Lin-DMA and PEG-C-DMA and distearoylphosphatidylcholine (DSPC), cholesterol and siRNA. The size of the resulting SNALP liposomes can be about 80-100 nm. SNALP can include synthetic cholesterol (Sigma-Aldrich, St Louis, Mo., USA), dipalmitoylphosphatidylcholine (AvantiPolar Lipids, Alabaster, Ala., USA), 3-N-[(w-methoxy poly (ethylene glycol) 2000) carbamoyl]-1,2-dimyristoyloxypropylamine and cationic 1,2-dilinoleyloxy-3-N, N dimethylaminopropane. SNALP may comprise synthetic cholesterol (Sigma-Aldrich), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC; AvantiPolar Lipids Inc.), PEG-cDMA, and 1,2-dilinoleyloxy-3-(N;N-dimethyl)aminopropane (DLinDMA).

[0296] Other cationic lipids such as amino lipid 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA) can be used to deliver CasZ polypeptides of the present disclosure, CasZ fusion polypeptides of the present disclosure, RNPs of the present disclosure, nucleic acids of the present disclosure (e.g., CasZ guide RNA and / or CasZ trancRNA) or CasZ systems of the present disclosure to target cells. Preformed vesicles with the following lipid composition can be considered: amino lipids, distearoylphosphatidylcholine (DSPC), cholesterol, and (R)-2,3-bis(octadecyloxy)propyl-1-(methoxypoly(ethylene glycol) 2000)propylcarbamate (PEG-lipid) in a molar ratio of 40 / 10 / 40 / 10, respectively, and a FVIIsiRNA / total lipid ratio of about 0.05 (w / w). To ensure a narrow particle size distribution in the range of 70-90 nm and a low polydispersity index of 0.11.+-.0.04 (n=56), the particles can be extruded through an 80 nm membrane up to three times before adding the guide RNA. Particles containing the highly potent amino lipid 16 can be used, where the molar ratio of the four lipid components 16, DSPC, cholesterol, and PEG-lipid (50 / 10 / 38.5 / 1.5) can be further optimized to enhance in vivo activity.

[0297] Lipids can be formulated with the CasZ system of the present disclosure or one or more components thereof or nucleic acids encoding them to form lipid nanoparticles (LNPs). Suitable lipids include, but are not limited to, DLin-KC2-DMA4, C12-200 and colipids distearoylphosphatidylcholine, cholesterol and PEG-DMG. The CasZ system of the present disclosure or its components can be formulated using a spontaneous vesicle formation procedure. The component molar ratio can be about 50 / 10 / 38.5 / 1.5 (DLin-KC2-DMA or C12-200 / distearoylphosphatidylcholine / cholesterol / PEG-DMG).

[0298] The CasZ system of the present disclosure or components thereof can be delivered encapsulated in PLGA microspheres, such as those further described in U.S. Published Applications 20130252281, 20130245107, and 20130244279.

[0299] Supercharged proteins can be used to deliver CasZ polypeptides of the present disclosure, CasZ fusion polypeptides of the present disclosure, RNPs of the present disclosure, nucleic acids of the present disclosure (e.g., CasZ guide RNA and / or CasZ trancRNA) or CasZ systems of the present disclosure to target cells. Supercharged proteins are a class of engineered or naturally occurring proteins that have an abnormally high positive or negative net theoretical charge. Supernegatively charged proteins and superpositively charged proteins both show the ability to tolerate thermally or chemically induced aggregation. Superpositively charged proteins are also able to penetrate mammalian cells. Associating cargo with these proteins (such as plasmid DNA, RNA or other proteins) can achieve functional delivery of these macromolecules to mammalian cells in vitro and in vivo.

[0300] Cell penetrating peptides (CPPs) can be used to deliver CasZ polypeptides of the present disclosure, CasZ fusion polypeptides of the present disclosure, RNPs of the present disclosure, nucleic acids of the present disclosure (e.g., CasZ guide RNA and / or CasZ trancRNA) or CasZ systems of the present disclosure to target cells. CPPs typically have an amino acid composition containing high relative abundance of positively charged amino acids (such as lysine or arginine), or a sequence containing an alternating pattern of polar / charged amino acids and non-polar hydrophobic amino acids.

[0301] An implantable device can be used to deliver the CasZ polypeptide of the present invention, the CasZ fusion polypeptide of the present invention, the RNP of the present invention, the nucleic acid of the present invention (e.g., CasZ guide RNA and / or CasZ trancRNA) (e.g., CasZ guide RNA, nucleic acid encoding CasZ guide RNA, nucleic acid encoding CasZ polypeptide, donor template, etc.) or the CasZ system of the present invention to a target cell (e.g., a target cell in vivo, wherein the target cell is a target cell in circulation, a target cell in a tissue, a target cell in an organ, etc.). An implantable device suitable for delivering the CasZ polypeptide of the present invention, the CasZ fusion polypeptide of the present invention, the RNP of the present invention, the nucleic acid of the present invention (e.g., CasZ guide RNA and / or CasZ trancRNA) or the CasZ system of the present invention to a target cell (e.g., a target cell in vivo, wherein the target cell is a target cell in circulation, a target cell in a tissue, a target cell in an organ, etc.) can include a container (e.g., a reservoir, a matrix, etc.), wherein the container comprises a CasZ polypeptide, a CasZ fusion polypeptide, an RNP or a CasZ system (or a component thereof, such as a nucleic acid of the present invention).

[0302] Suitable implantable device can include, for example, a polymer substrate (such as matrix) used as a device body, and includes other support materials (such as metal or other polymers) in some cases, and enhances visibility and imaging materials. Implantable delivery device can be conducive to providing release locally and for a long time, wherein polypeptide to be delivered and / or nucleic acid are directly released to target sites, such as extracellular matrix (ECM), vascular system around tumors, diseased tissue etc. Suitable implantable delivery device includes a device suitable for delivery to cavity (such as peritoneal cavity) and / or any other type of administration that drug delivery system is not anchored or attached, and the device includes biostable and / or degradable and / or bioabsorbable polymer substrates, which can, for example, optionally be matrix. In some cases, suitable implantable drug delivery device includes degradable polymers, wherein main release mechanism is overall erosion (bulk erosion). In some cases, suitable implantable drug delivery devices include non-degradable or slowly degradable polymers, wherein the main release mechanism is diffusion rather than overall erosion, so that the outer portion is used as a film and its inner portion is used as a drug reservoir, in fact, the drug reservoir is not affected by the surrounding environment for a long time (e.g., about one week to about several months). The combination of different polymers with different release mechanisms can also be optionally used. During the effective period of the total release period, the concentration gradient can remain effectively constant, and therefore the diffusion rate is effectively constant (referred to as "zero mode" diffusion). The term "constant" means that the diffusion rate maintains a lower threshold value higher than the therapeutic effectiveness, but it is still optionally characterized by initial burst and / or can fluctuate, such as increase and decrease to a certain extent. Diffusion rate can be maintained for a long time, and it can be considered that the diffusion rate is constant to a certain level to optimize the therapeutic effective period, such as an effective silent period.

[0303] In some cases, the implantable delivery system is designed to protect the nucleotide-based therapeutic from degradation, either chemically or due to attack by enzymes and other factors within the subject's body.

[0304] The implantation site or target site of the device can be selected for maximum therapeutic efficacy. For example, the delivery device can be implanted within or near the tumor environment, or within or near the blood supply associated with the tumor. Target locations can be, for example: 1) brain degeneration sites, such as in Parkinson's disease or Alzheimer's disease at the basal ganglia, white matter and gray matter; 2) spine, such as in amyotrophic lateral sclerosis (ALS); 3) cervix; 4) active and chronic inflammatory joints; 5) dermis, such as in psoriasis; 7) sympathetic and sensory nerve sites for analgesia; 7) bone; 8) acute or chronic infection sites; 9) intravaginal; 10) inner ear - auditory system, inner ear labyrinth, vestibular system; 11) intratracheal; 12) intracardiac; coronary arteries, epicardium; 13) urinary tract or bladder; 14) biliary system; 15) parenchymal tissue, including but not limited to kidney, liver, spleen; 16) lymph nodes; 17) salivary glands; 18) gums; 19) intraarticular (into a joint); 20) intraocular; 21) brain tissue; 22) brain ventricles; 23) cavities, including the abdominal cavity (for example but not limited to ovarian cancer); 24) within the esophagus; and 25) within the rectum; and 26) into the vasculature.

[0305] Insertion methods (such as implantation) may optionally already be used for other types of tissue implantation and / or for insertion and / or for tissue sampling, optionally without modification, or alternatively only with optional non-major modifications in such methods. Such methods optionally include, but are not limited to, brachytherapy methods, biopsy, endoscopy with and / or without ultrasound (such as stereotactic methods to access brain tissue), laparoscopy (including implantation with a laparoscope into joints, abdominal organs, bladder walls, and body cavities).

[0306] Modified host cells

[0307] The present disclosure provides a modified cell, the modified cell comprises a CasZ polypeptide of the present disclosure and / or a nucleic acid comprising a nucleotide sequence encoding a CasZ polypeptide of the present disclosure. The present disclosure provides a modified cell, the modified cell comprises a CasZ polypeptide of the present disclosure, wherein the modified cell is a cell that does not generally comprise a CasZ polypeptide of the present disclosure. The present disclosure provides a modified cell (e.g., a genetically modified cell), the modified cell comprises a nucleic acid, the nucleic acid comprises a nucleotide sequence encoding a CasZ polypeptide of the present disclosure. The present disclosure provides a genetically modified cell genetically modified with mRNA, the mRNA comprising a nucleotide sequence encoding a CasZ polypeptide of the present disclosure. The present disclosure provides a genetically modified cell genetically modified with a recombinant expression vector, the recombinant expression vector comprising a nucleotide sequence encoding a CasZ polypeptide of the present disclosure. The present disclosure provides a genetically modified cell genetically modified with a recombinant expression vector, the recombinant expression vector comprising: a) a nucleotide sequence encoding a CasZ polypeptide of the present disclosure; and b) a nucleotide sequence encoding a CasZ guide RNA of the present disclosure. The present disclosure provides a genetically modified cell genetically modified with a recombinant expression vector, the recombinant expression vector comprising: a) a nucleotide sequence encoding a CasZ polypeptide of the present disclosure; b) a nucleotide sequence encoding a CasZ guide RNA of the present disclosure; and c) a nucleotide sequence encoding a donor template.

[0308] Cells used as receptors for CasZ polypeptides of the present invention and / or nucleic acids comprising nucleotide sequences encoding CasZ polypeptides of the present invention and / or CasZ guide RNAs of the present invention (or nucleic acids encoding it) and / or CasZ trancRNAs (or nucleic acids encoding it) can be any of a variety of cells, including, for example, in vitro cells; in vivo cells; ex vivo cells; primary cells; cancer cells; animal cells; plant cells; algae cells; fungal cells, etc. Cells used as receptors for CasZ polypeptides of the present invention and / or nucleic acids comprising nucleotide sequences encoding CasZ polypeptides of the present invention and / or CasZ guide RNAs of the present invention are referred to as "host cells" or "target cells". Host cells or target cells can be receptors for the CasZ system of the present invention. Host cells or target cells can be receptors for CasZ RNPs of the present invention. Host cells or target cells can be receptors for a single component of the CasZ system of the present invention.

[0309] Non-limiting examples of cells (target cells) include: prokaryotic cells, eukaryotic cells, bacterial cells, archaeal cells, cells of unicellular eukaryotic organisms, protozoan cells, cells from plants (e.g., cells from plant crops, fruits, vegetables, grains, soybeans, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugar cane, pumpkin, hay, potatoes, cotton, hemp, tobacco, flowering plants, conifers, gymnosperms, angiosperms, ferns, lycophytes, hornworts, liverworts, mosses, dicots, monocots, etc.), algal cells (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum spratense, etc.), patens), feather algae (C. agardh), etc.), seaweed (e.g., giant kelp), fungal cells (e.g., yeast cells, cells from mushrooms), animal cells, cells from invertebrates (e.g., fruit flies, cnidarians, echinoderms, nematodes, etc.), cells from vertebrates (e.g., fish, amphibians, reptiles, birds, mammals), cells from mammals (e.g., ungulates (e.g., pigs, cows, goats, sheep); rodents (e.g., rats, mice); non-human primates; humans; felines (e.g., cats); canines (e.g., dogs), etc.), etc. In some cases, the cell is a cell that is not derived from a natural organism (e.g., the cell can be a synthetically made cell; also referred to as an artificial cell).

[0310] The cell may be an in vitro cell (e.g., a cell in culture, such as an established cultured cell line). The cell may be an ex vivo cell (a cultured cell from an individual). The cell may be an in vivo cell (e.g., a cell in an individual). The cell may be an isolated cell. The cell may be a cell inside an organism. The cell may be an organism. The cell may be a cell in a cell culture (e.g., an in vitro cell culture). The cell may be one of a collection of cells. The cell may be a prokaryotic cell or derived from a prokaryotic cell. The cell may be a bacterial cell or may be derived from a bacterial cell. The cell may be an archaeal cell or may be derived from an archaeal cell. The cell may be a eukaryotic cell or may be derived from a eukaryotic cell. The cell may be a plant cell or may be derived from a plant cell. The cell may be an animal cell or may be derived from an animal cell. The cell may be an invertebrate cell or may be derived from an invertebrate cell. The cell may be a vertebrate cell or may be derived from a vertebrate cell. The cell may be a mammalian cell or may be derived from a mammalian cell. The cell may be a rodent cell or may be derived from a rodent cell. The cell may be a human cell or may be derived from a human cell. The cell may be a microbial cell or may be derived from a microbial cell. The cell may be a fungal cell or derived from a fungal cell. The cell may be an insect cell. The cell may be an arthropod cell. The cell may be a protozoan cell. The cell may be a worm cell.

[0311] Suitable cells include stem cells (e.g., embryonic stem (ES) cells, induced pluripotent stem (iPS) cells; germ cells (e.g., oocytes, sperm, oogonia, spermatogonia, etc.); somatic cells, such as fibroblasts, oligodendrocytes, glial cells, hematopoietic cells, neurons, muscle cells, bone cells, hepatocytes, pancreatic cells, etc.

[0312] Suitable cells include human embryonic stem cells, embryonic cardiomyocytes, myofibroblasts, mesenchymal stem cells, autologous expanded cardiomyocytes, adipocytes, totipotent cells, pluripotent cells, blood stem cells, myoblasts, adult stem cells, bone marrow cells, mesenchymal cells, embryonic stem cells, parenchymal cells, epithelial cells, endothelial cells, mesothelial cells, fibroblasts, osteoblasts, chondrocytes, exogenous cells, endogenous cells, stem cells, hematopoietic stem cells, bone marrow-derived progenitor cells, cardiomyocytes, skeletal cells, fetal cells, undifferentiated cells, multipotent progenitor cells, unipotent progenitor cells, monocytes, cardiac myoblasts, skeletal myoblasts, macrophages, capillary endothelial cells, xenogeneic cells, allogeneic cells, and postpartum stem cells.

[0313] In some cases, the cell is an immune cell, a neuron, an epithelial cell and an endothelial cell or a stem cell. In some cases, the immune cell is a T cell, a B cell, a monocyte, a natural killer cell, a dendritic cell or a macrophage. In some cases, the immune cell is a cytotoxic T cell. In some cases, the immune cell is a helper T cell. In some cases, the immune cell is a regulatory T cell (Treg).

[0314] In some cases, the cell is a stem cell. Stem cells include adult stem cells. Adult stem cells are also called somatic stem cells.

[0315] Adult stem cells reside in differentiated tissues but retain the properties of self-renewal and the ability to generate a variety of cell types, often typical of the tissue in which the stem cell resides. Many examples of somatic stem cells are known to those skilled in the art, including muscle stem cells; hematopoietic stem cells; epithelial stem cells; neural stem cells; mesenchymal stem cells; mammary stem cells; intestinal stem cells; mesoderm stem cells; endothelial stem cells; olfactory stem cells; neural crest stem cells, etc.

[0316] Target stem cells include mammalian stem cells, wherein the term "mammal" refers to any animal classified as a mammal, including humans; non-human primates; livestock and farm animals; and zoo, laboratory, sports or pet animals, such as dogs, horses, cats, cows, mice, rats, rabbits, etc. In some cases, the stem cells are human stem cells. In some cases, the stem cells are rodent (e.g., mouse; rat) stem cells. In some cases, the stem cells are non-human primate stem cells.

[0317] The stem cells may express one or more stem cell markers, such as SOX9, KRT19, KRT7, LGR5, CA9, FXYD2, CDH6, CLDN18, TSPAN8, BPIFB1, OLFM4, CDH17, and PPARGC1A.

[0318] In some cases, the stem cell is a hematopoietic stem cell (HSC). HSCs are mesoderm-derived cells that can be isolated from bone marrow, blood, umbilical cord blood, fetal liver, and yolk sac. HSCs are characterized by CD34 + and CD3 - HSCs can regenerate erythroid, neutrophil-macrophage, megakaryocyte, and lymphoid hematopoietic cell lineages in vivo. In vitro, HSCs can be induced to undergo at least some self-renewing cell divisions, and can be induced to differentiate into the same lineages as seen in vivo. Thus, HSCs can be induced to differentiate into one or more of erythroid, megakaryocyte, neutrophil, macrophage, and lymphocyte.

[0319] In other cases, the stem cell is a neural stem cell (NSC). Neural stem cells (NSC) can differentiate into neurons and glial cells (including oligodendrocytes and astrocytes). Neural stem cells are multipotent stem cells that can divide multiple times, and under certain conditions, can produce daughter cells as neural stem cells, or can be used as neural progenitor cells of neuroblasts or glial cells, for example, cells that are committed to becoming neurons and glial cells of one or more types, respectively. Methods for obtaining NSC are known in the art.

[0320] In other cases, the stem cell is a mesenchymal stem cell (MSC). MSCs are originally derived from embryonic mesoderm and isolated from adult bone marrow and can differentiate into muscle, bone, cartilage, fat, bone marrow stroma, and tendon. Methods for isolating MSCs are known in the art; and any known method can be used to obtain MSCs. See, for example, U.S. Patent No. 5,736,396, which describes the isolation of human MSCs.

[0321] In some cases, the cell is a plant cell. The plant cell can be a cell of a monocot. The cell can be a cell of a dicot.

[0322] In some cases, the cell is a plant cell. For example, the cell can be a cell of a major agricultural plant, such as barley, beans (dry edible), rapeseed, corn, cotton (Pima cotton), cotton (Upland cotton), linseed, hay (alfalfa), hay (non-alfalfa), oats, peanuts, rice, sorghum, soybeans, sugar beets, sugar cane, sunflower (oil), sunflower (non-oil), sweet potato, tobacco (burley), tobacco (flue-cured), tomato, wheat (durum wheat), wheat (spring wheat), wheat (winter wheat), etc. As another example, the cell is a cell of a vegetable crop, which includes, but is not limited to, for example, alfalfa sprouts, aloe vera leaves, arrowroot, arrowhead, artichoke, asparagus, bamboo shoots, banana flowers, bean sprouts, beans, beet greens, beets, bitter melon, cabbage, broccoli, Brussels sprouts, Brussels sprouts, cabbage, cabbage sprouts, cactus leaves (cactus fruit), winter squash, spiny thistle, carrot, cauliflower, celery, chayote, Chinese artichoke (crosne), Chinese cabbage, Chinese celery, Chinese leek, Chinese vegetable heart, chrysanthemum leaves (tung ho), kale, corn stover, sweet corn, cucumber, daikon, dandelion greens, dasheen, dau mue (pea tip), donqua (winter squash), eggplant, endive, lettuce, fiddlehead fern, field cress, endive, mustard greens, gailon, galangal (Siamese, Thai ginger), garlic, ginger root, burdock (gobo), young leaves, Hanover salad greens, huauzontle, Jerusalem artichoke, jicama, young kale leaves, kohlrabi, quilete, lettuce (bibb), lettuce (boston), lettuce (boston red), lettuce (green leaf), lettuce (iceberg), lettuce (lolla rossa), lettuce (green oak leaf), lettuce (red oak leaf), lettuce (processed lettuce), lettuce (red leaf), lettuce (romaine), lettuce (ruby romaine), lettuce (Russian red mustard), linkok, white radish (lo bok), long beans, lotus root, lettuce (mache), agave (agave) leaves, malanga, mesculin mix, mizuna, moap (smooth loofah), moo, moqua(fuzzy squash), mushrooms, mustard, yams (nagaimo), okra, water spinach, onion greens, opo (long squash), ornamental corn, ornamental gourds, parsley, parsnips, peas, peppers (bell-shaped), peppers, pumpkin (pumpkin), endive (radicchio), radish sprouts, radishes (radish), brassica oleracea, brassica oleracea, rhubarb, romaine lettuce (baby red), rutabaga, salsa verde (sea bean), luffa (horn / ridged), spinach, squash, straw bales, sugar cane, sweet potato, Swiss chard, tamarind, taro, taro leaves, taro sprouts, taro leaf, tepeguaje (gourd), tindora, tomatillo, tomato, cherry, grape, plum, turmeric, turnip stems and leaves, turnip, water chestnut, yampi, yam (name), yu choy, yuca (cassava), etc.

[0323] In some cases, the cell is an arthropod cell. For example, the cell can be a cell of a suborder, family, subfamily, group, subpopulation, or species such as Chelicerata, Myriapodia, Hexipodia, Arachnida, Insecta, Archaeognatha, Thysanura, Palaeoptera, Ephemeroptera, Odonata, Anisoptera, Zygoptera, Neoptera, Exopterygota, Plecoptera, Embioptera, Orthoptera, Zoraptera, Dermaptera, Dictyoptera, Notoptera, Gryllotalpa, and Lycoptera. loblattidae, Mantophasmatidae, Phasmatodea, Blattaria, Isoptera, Mantodea, Parapneuroptera, Psocoptera, Thysanoptera, Phthiraptera, Hemiptera, Endopterygota or Holometabola, Hymenoptera, Coleoptera, Strepsiptera, Raphidioptera, Megaloptera, Neuroptera, Mecoptera, Siphonaptera, Diptera, Trichoptera or Lepidoptera.

[0324] In some cases, the cell is an insect cell. For example, in some cases, the cell is a cell of a mosquito, grasshopper, hemiptera, fly, flea, bee, wasp, ant, louse, moth, or beetle.

[0325] Reagent test kit

[0326] The present disclosure provides a kit comprising the CasZ system of the present disclosure or a component of the CasZ system of the present disclosure.

[0327] The kit of the present disclosure may include any combination as listed for the CasZ system (e.g., see above). The kit of the present disclosure may include: a) components of the CasZ system of the present disclosure as described above, or may include the CasZ system of the present disclosure; and b) one or more additional reagents, for example, i) buffers; ii) protease inhibitors; iii) nuclease inhibitors; iv) reagents required for developing or visualizing detectable labels; v) positive and / or negative control target DNA; vi) positive and / or negative control CasZ guide RNA; vii) CasZ trancRNA, etc. The kit of the present disclosure may include: a) components of the CasZ system of the present disclosure as described above, or may include the CasZ system of the present disclosure; and b) therapeutic agents.

[0328] The kit of the present disclosure may include a recombinant expression vector, comprising: a) an insertion site for inserting a nucleic acid, the nucleic acid comprising a nucleotide sequence encoding a portion of a CasZ guide RNA, a portion of which hybridizes with a target nucleotide sequence in a target nucleic acid; and b) a nucleotide sequence encoding a CasZ binding portion of a CasZ guide RNA. The kit of the present disclosure may include a recombinant expression vector, the recombinant expression vector comprising: a) an insertion site for inserting a nucleic acid, the nucleic acid comprising a nucleotide sequence encoding a portion of a CasZ guide RNA, a portion of which hybridizes with a target nucleotide sequence in a target nucleic acid; b) a nucleotide sequence encoding a CasZ binding portion of a CasZ guide RNA; and c) a nucleotide sequence encoding a CasZ polypeptide of the present disclosure. The kit of the present disclosure may include a recombinant expression vector, the recombinant expression vector comprising a nucleotide sequence encoding a CasZ trancRNA.

[0329] Detection of SSDNA

[0330] Once the CasZ (Cas14) polypeptide of the present disclosure is activated by detecting target DNA (double-stranded or single-stranded), it will promiscuously cut non-targeted single-stranded DNA (ssDNA). Once CasZ (Cas14) is activated by guide RNA (when the guide RNA hybridizes with the target sequence of the target DNA (that is, the sample includes target DNA, such as target ssDNA)), the protein will become a nuclease that promiscuously cuts ssDNA (that is, the nuclease cuts non-target ssDNA, that is, the guide sequence of the guide RNA is not hybridized with ssDNA). Therefore, when the target DNA is present in the sample (for example, in some cases exceeding the threshold amount), the cutting of ssDNA in the sample is caused, which can be detected using any convenient detection method (for example, using labeled single-stranded detection agent DNA). In some cases, in addition to the CasZ guide RNA, the CasZ polypeptide also requires tranc RNA for activation.

[0331] Provided are compositions and methods for detecting a target DNA (double-stranded or single-stranded) in a sample. In some cases, a detector DNA that is single-stranded (ssDNA) and does not hybridize with the guide sequence of a guide RNA is used (i.e., the detector ssDNA is a non-target ssDNA). Such methods may include (a) contacting a sample with the following substances: (i) a CasZ polypeptide; (ii) a guide RNA, the guide RNA comprising a region bound to the CasZ polypeptide and a guide sequence hybridized with the target DNA; and (iii) a detector DNA that is single-stranded and does not hybridize with the guide sequence of the guide RNA; and (b) measuring a detectable signal generated by cleavage of the single-stranded detector DNA by the CasZ polypeptide, thereby detecting the target DNA. In some cases, the method may include (a) contacting the sample with the following substances: (i) CasZ polypeptide; (ii) guide RNA, the guide RNA comprising a region bound to the CasZ polypeptide and a guide sequence hybridized with the target DNA; (iii) CasZ tranc RNA; and (iv) a detector DNA that is single-stranded and does not hybridize with the guide sequence of the guide RNA; and (b) measuring the detectable signal generated by the CasZ polypeptide cutting the single-stranded detector DNA, thereby detecting the target DNA. As noted above, once the subject CasZ polypeptide protein is activated by the guide RNA (occurring when the sample contains a target DNA hybridized with the guide RNA (i.e., the sample contains a targeted target DNA)), the CasZ polypeptide is activated and acts as an endoribonuclease to non-specifically cut the ssDNA (including non-target ssDNA) present in the sample. Therefore, when the targeted target DNA is present in the sample (e.g., in some cases exceeding a threshold amount), the cutting of ssDNA (including non-target ssDNA) in the sample is caused, which can be detected using any convenient detection method (e.g., using a labeled detector ssDNA).

[0332] Also provided are compositions and methods for cutting single-stranded DNA (ssDNA) (e.g., non-target ssDNA). Such methods may include contacting a nucleic acid population with the following material, wherein the population comprises a target DNA and multiple non-target ssDNAs: (i) a CasZ polypeptide; and (ii) a guide RNA, wherein the guide RNA comprises a region bound to the CasZ polypeptide and a guide sequence hybridized with the target DNA, wherein the CasZ polypeptide cuts the multiple non-target ssDNAs. Such methods may include contacting a nucleic acid population with the following material, wherein the population comprises a target DNA and multiple non-target ssDNAs: (i) a CasZ polypeptide; (ii) a guide RNA, wherein the guide RNA comprises a region bound to the CasZ polypeptide and a guide sequence hybridized with the target DNA; and (iii) a CasZ tranc RNA, wherein the CasZ polypeptide cuts the multiple non-target ssDNAs. Such methods may be used, for example, to cut exogenous ssDNA (e.g., viral DNA) in a cell.

[0333] The contacting step of the subject method can be performed in a composition comprising a divalent metal ion. The contacting step can be performed in a cell-free environment, such as outside a cell. The contacting step can be performed inside a cell. The contacting step can be performed in an in vitro cell. The contacting step can be performed in an ex vivo cell. The contacting step can be performed in an in vivo cell.

[0334] The guide RNA can be provided in the form of RNA or in the form of a nucleic acid encoding the guide RNA (e.g., DNA, such as a recombinant expression vector). The tranc RNA can be provided in the form of RNA or in the form of a nucleic acid encoding the guide RNA (e.g., DNA, such as a recombinant expression vector). The CasZ polypeptide can be provided as a protein itself or in the form of a nucleic acid encoding the protein (e.g., mRNA, DNA such as a recombinant expression vector). In some cases, two or more (e.g., 3 or more, 4 or more, 5 or more or 6 or more) guide RNAs can be provided. In some cases, a single molecule RNA is used, comprising: i) CasZ guide RNA; and ii) tranc RNA (or a nucleic acid comprising a nucleotide sequence encoding a single molecule RNA).

[0335] In some cases (e.g., when the sample is contacted with a guide RNA and a CasZ polypeptide; or when the sample is contacted with a guide RNA, a CasZ polypeptide, and a tranc RNA), the sample is contacted for 2 hours or less (e.g., 1.5 hours or less, 1 hour or less, 40 minutes or less, 30 minutes or less, 20 minutes or less, 10 minutes or less, or 5 minutes or less, or 1 minute or less) before the measuring step. For example, in some cases, the sample is contacted for 40 minutes or less before the measuring step. In some cases, the sample is contacted for 20 minutes or less before the measuring step. In some cases, the sample is contacted for 10 minutes or less before the measuring step. In some cases, the sample is contacted for 5 minutes or less before the measuring step. In some cases, the sample is contacted for 1 minute or less before the measuring step. In some cases, the sample is contacted for 50 to 60 seconds before the measuring step. In some cases, the sample is contacted for 40 seconds to 50 seconds prior to the measuring step. In some cases, the sample is contacted for 30 seconds to 40 seconds prior to the measuring step. In some cases, the sample is contacted for 20 seconds to 30 seconds prior to the measuring step. In some cases, the sample is contacted for 10 seconds to 20 seconds prior to the measuring step.

[0336] In some cases, the method for detecting target DNA disclosed herein comprises: a) contacting a sample with a guide RNA, a CasZ polypeptide, and a detector DNA under conditions that provide trans-cleavage of the detector DNA, wherein the sample is contacted for 2 hours or less (e.g., 1.5 hours or less, 1 hour or less, 40 minutes or less, 30 minutes or less, 20 minutes or less, 10 minutes or less, or 5 minutes or less, or 1 minute or less); b) maintaining the sample from step (a) for a period of time under conditions that do not provide trans-cleavage of the detector RNA; and c) after the period of time in step (b), measuring a detectable signal generated by cleavage of the single-stranded detector DNA by the CasZ polypeptide, thereby detecting the target DNA. The conditions that provide trans-cleavage of the detector DNA include temperature conditions, such as from 17° C. to about 39° C. (e.g., about 37° C.). Conditions that do not provide for trans-cleavage of the detector DNA include temperatures of 10°C or less, 5°C or less, 4°C or less, or 0°C.

[0337] In some cases, the method for detecting target DNA of the present disclosure comprises: a) contacting a sample with a guide RNA, a tranc RNA, a CasZ polypeptide, and a detector DNA (or contacting the sample with: i) a single-molecule RNA comprising a guide RNA and a tranc RNA; i) a CasZ polypeptide; and iii) a detector DNA) under conditions that provide trans-cleavage of the detector DNA, wherein the sample is contacted for 2 hours or less (e.g., 1.5 hours or less, 1 hour or less, 40 minutes or less, 30 minutes or less, 20 minutes or less, 10 minutes or less, or 5 minutes or less, or 1 minute or less); b) maintaining the sample from step (a) for a period of time under conditions that do not provide trans-cleavage of the detector RNA; and c) after the period of time in step (b), measuring a detectable signal generated by cleavage of the single-stranded detector DNA by the CasZ polypeptide, thereby detecting the target DNA. Conditions that provide trans-cleavage of the detector DNA include temperature conditions such as from 17°C to about 39°C (e.g., about 37°C). Conditions that do not provide trans-cleavage of the detector DNA include temperatures of 10°C or less, 5°C or less, 4°C or less, or 0°C.

[0338] In some cases, the generation time of the detectable signal generated by cutting the single-stranded detector DNA is no more than 60 minutes. For example, in some cases, the generation time of the detectable signal generated by cutting the single-stranded detector DNA is no more than 60 minutes, no more than 45 minutes, no more than 30 minutes, no more than 15 minutes, no more than 10 minutes or no more than 5 minutes. For example, in some cases, the generation time period of the detectable signal generated by cutting the single-stranded detector DNA is 1 minute to 60 minutes, such as 1 minute to 5 minutes, 5 minutes to 10 minutes, 10 minutes to 15 minutes, 15 minutes to 30 minutes, 30 minutes to 45 minutes or 45 minutes to 60 minutes. In some cases, after the generation of the detectable signal (for example, the generation time is no more than 60 minutes), the generation of the detection signal can be stopped by the following methods, for example, reducing the temperature of the sample (for example, reducing the temperature to 10°C or lower, 5°C or lower, 4°C or lower or 0°C), adding an inhibitor to the sample, freeze-drying the sample, heating the sample to above 40°C, etc. The measuring step can be performed at any time after the generation of the detectable signal has been stopped. For example, the measuring step can be performed 5 minutes to 48 hours after the production of the detectable signal has ceased. For example, the measuring step can be performed 5 minutes to 15 minutes, 15 minutes to 30 minutes, 30 minutes to 60 minutes, 1 hour to 4 hours, 4 hours to 8 hours, 8 hours to 12 hours, 12 hours to 24 hours, 24 hours to 36 hours, or 36 hours to 48 hours after the production of the detectable signal has ceased. The measuring step can be performed more than 48 hours after the production of the detectable signal has ceased.

[0339] The method for detecting a target DNA (single-stranded or double-stranded) in a sample disclosed herein can detect the target DNA with high sensitivity. In some cases, the method disclosed herein can be used to detect a target DNA present in a sample containing a plurality of DNAs (including a target DNA and a plurality of non-target DNAs), wherein the target DNA is present at a concentration of 10 7 One or more copies of non-target DNA (e.g., 6 One or more copies of non-target DNA, every 10 5 One or more copies of non-target DNA, every 10 4 One or more copies of non-target DNA, every 10 3 One or more copies of non-target DNA, every 10 2 In some cases, the methods of the present disclosure can be used to detect a target DNA present in a sample comprising a plurality of DNAs (including a target DNA and a plurality of non-target DNAs), wherein the target DNA is present at a rate of 10 copies per 10 non-target DNAs, 10 copies per 10 non-target DNAs, 10 copies per 10 non-target DNAs, 10 copies per 10 non-target DNAs, or 10 copies per 10 non-target DNAs. 18One or more copies of non-target DNA (e.g., 15 One or more copies of non-target DNA, every 10 12 One or more copies of non-target DNA, every 10 9 One or more copies of non-target DNA, every 10 6 One or more copies of non-target DNA, every 10 5 One or more copies of non-target DNA, every 10 4 One or more copies of non-target DNA, every 10 3 One or more copies of non-target DNA, every 10 2 non-target DNA, one or more copies per 50 non-target DNAs, one or more copies per 20 non-target DNAs, one or more copies per 10 non-target DNAs, or one or more copies per 5 non-target DNAs).

[0340] In some cases, the methods of the present disclosure can detect target DNA present in a sample, wherein the target DNA is present at a concentration of 10 7 One copy per 10 non-target DNA to one copy per 10 non-target DNA (e.g., 7 1 copy of non-target DNA to 10 2 1 copy of non-target DNA, 10 7 1 copy of non-target DNA to 10 3 1 copy of non-target DNA, 10 7 1 copy of non-target DNA to 10 4 1 copy of non-target DNA, 10 7 1 copy of non-target DNA to every 10 5 1 copy of non-target DNA, 10 7 1 copy of non-target DNA to 10 6 1 copy of non-target DNA, 10 6 1 copy per 10 non-target DNA to 1 copy per 10 non-target DNA, 6 1 copy of non-target DNA to 10 2 1 copy of non-target DNA, 10 6 1 copy of non-target DNA to 10 3 1 copy of non-target DNA, 10 6 1 copy of non-target DNA to 10 4 1 copy of non-target DNA, 10 6 1 copy of non-target DNA to 10 5 1 copy of non-target DNA, 10 51 copy per 10 non-target DNA to 1 copy per 10 non-target DNA, 5 1 copy of non-target DNA to 10 2 1 copy of non-target DNA, 10 5 1 copy of non-target DNA to 10 3 1 copy of non-target DNA, or 10 5 1 copy of non-target DNA to 10 4 1 copy of non-target DNA) is present.

[0341] In some cases, the methods of the present disclosure can detect target DNA present in a sample, wherein the target DNA is present at a concentration of 10 18 One copy per 10 non-target DNA to one copy per 10 non-target DNA (e.g., 18 1 copy of non-target DNA to 10 2 1 copy of non-target DNA, 10 15 1 copy of non-target DNA to 10 2 1 copy of non-target DNA, 10 12 1 copy of non-target DNA to 10 2 1 copy of non-target DNA, 10 9 1 copy of non-target DNA to 10 2 1 copy of non-target DNA, 10 7 1 copy of non-target DNA to every 10 2 1 copy of non-target DNA, 10 7 1 copy of non-target DNA to 10 3 1 copy of non-target DNA, 10 7 1 copy of non-target DNA to 10 4 1 copy of non-target DNA, 10 7 1 copy of non-target DNA to 10 5 1 copy of non-target DNA, 10 7 1 copy of non-target DNA to 10 6 1 copy of non-target DNA, 10 6 1 copy per 10 non-target DNA to 1 copy per 10 non-target DNA, 6 1 copy of non-target DNA to 10 2 1 copy of non-target DNA, 10 6 1 copy of non-target DNA to every 10 3 1 copy of non-target DNA, 10 6 1 copy of non-target DNA to 10 4 1 copy of non-target DNA, 106 1 copy of non-target DNA to 10 5 1 copy of non-target DNA, 10 5 1 copy per 10 non-target DNA to 1 copy per 10 non-target DNA, 5 1 copy of non-target DNA to 10 2 1 copy of non-target DNA, 10 5 1 copy of non-target DNA to 10 3 1 copy of non-target DNA, or 10 5 1 copy of non-target DNA to 10 4 1 copy of non-target DNA) is present.

[0342] In some cases, the methods of the present disclosure can detect target DNA present in a sample, wherein the target DNA is present at a concentration of 10 7 One copy per 100 non-target DNAs (e.g., one copy per 10 7 1 copy of non-target DNA to 10 2 1 copy of non-target DNA, 10 7 1 copy of non-target DNA to 10 3 1 copy of non-target DNA, 10 7 1 copy of non-target DNA to 10 4 1 copy of non-target DNA, 10 7 1 copy of non-target DNA to every 10 5 1 copy of non-target DNA, 10 7 1 copy of non-target DNA to 10 6 1 copy of non-target DNA, 10 6 1 copy per 100 non-target DNA, 1 copy per 10 6 1 copy of non-target DNA to 10 2 1 copy of non-target DNA, 10 6 1 copy of non-target DNA to 10 3 1 copy of non-target DNA, 10 6 1 copy of non-target DNA to 10 4 1 copy of non-target DNA, 10 6 1 copy of non-target DNA to 10 5 1 copy of non-target DNA, 10 5 1 copy per 100 non-target DNA, 1 copy per 10 5 1 copy of non-target DNA to 10 2 1 copy of non-target DNA, 105 1 copy of non-target DNA to 10 3 1 copy of non-target DNA, or 10 5 1 copy of non-target DNA to 10 4 1 copy of non-target DNA) is present.

[0343] In some cases, for the subject method of detecting the target DNA in the sample, the detection threshold is 10nM or less. Therefore, for example, the target DNA can be present in the sample at a concentration of 10nM or less. The term "detection threshold" is used herein to describe the minimum amount of target DNA that must be present in the sample for detection to occur. Therefore, as an illustrative example, when the detection threshold is 10nM, then when the target DNA is present in the sample at a concentration of 10nM or more, a signal can be detected. In some cases, the detection threshold of the method disclosed herein is 5nM or less. In some cases, the detection threshold of the method disclosed herein is 1nM or less. In some cases, the detection threshold of the method disclosed herein is 0.5nM or less. In some cases, the detection threshold of the method disclosed herein is 0.1nM or less. In some cases, the detection threshold of the method disclosed herein is 0.05nM or less. In some cases, the detection threshold of the method disclosed herein is 0.01nM or less. In some cases, the detection threshold of the method disclosed herein is 0.005nM or less. In some cases, the detection threshold of the method of the present disclosure is 0.001nM or less. In some cases, the detection threshold of the method of the present disclosure is 0.0005nM or less. In some cases, the detection threshold of the method of the present disclosure is 0.0001nM or less. In some cases, the detection threshold of the method of the present disclosure is 0.00005nM or less. In some cases, the detection threshold of the method of the present disclosure is 0.00001nM or less. In some cases, the detection threshold of the method of the present disclosure is 10pM or less. In some cases, the detection threshold of the method of the present disclosure is 1pM or less. In some cases, the detection threshold of the method of the present disclosure is 500fM or less. In some cases, the detection threshold of the method of the present disclosure is 250fM or less. In some cases, the detection threshold of the method of the present disclosure is 100fM or less. In some cases, the detection threshold of the method of the present disclosure is 50fM or less. In some cases, the detection threshold of the method of the present disclosure is 500aM (attomole) or less. In some cases, the detection threshold of the method of the present disclosure is 250aM or less. In some cases, the detection threshold of the method of the present disclosure is 100aM or less. In some cases, the detection threshold of the method of the present disclosure is 50aM or less. In some cases, the detection threshold of the method of the present disclosure is 10aM or less. In some cases, the detection threshold of the method of the present disclosure is 1aM or less.

[0344] In some cases, the detection threshold (for detecting target DNA with the subject methods) is in the range of 500fM to 1nM (e.g., 500fM to 500pM, 500fM to 200pM, 500fM to 100pM, 500fM to 10pM, 500fM to 1pM, 800fM to 1nM, 800fM to 500pM, 800fM to 200pM, 800fM to 100pM, 800fM to 10pM, 800fM to 1pM, 1pM to 1nM, 1pM to 500pM, 1pM to 200pM, 1pM to 100pM, or 1pM to 10pM) (where concentration refers to the threshold concentration of target DNA at which target DNA can be detected). In some cases, the detection threshold of the methods of the present disclosure is in the range of 800fM to 100pM. In some cases, the detection threshold of the methods of the present disclosure is in the range of 1 pM to 10 pM. In some cases, the detection threshold of the methods of the present disclosure is in the range of 10 fM to 500 fM, such as 10 fM to 50 fM, 50 fM to 100 fM, 100 fM to 250 fM, or 250 fM to 500 fM.

[0345] In some cases, the minimum concentration at which the target DNA can be detected in a sample is in the range of 500 fM to 1 nM (e.g., 500 fM to 500 pM, 500 fM to 200 pM, 500 fM to 100 pM, 500 fM to 10 pM, 500 fM to 1 pM, 800 fM to 1 nM, 800 fM to 500 pM, 800 fM to 200 pM, 800 fM to 100 pM, 800 fM to 10 pM, 800 fM to 1 pM, 1 pM to 1 nM, 1 pM to 500 pM, 1 pM to 200 pM, 1 pM to 100 pM, or 1 pM to 10 pM). In some cases, the minimum concentration at which the target DNA can be detected in a sample is in the range of 800 fM to 100 pM. In some cases, the minimum concentration at which target DNA can be detected in a sample is in the range of 1 pM to 10 pM.

[0346] In some cases, the detection threshold (for detecting a target DNA with the subject methods) is between 1aM and 1nM (e.g., 1aM to 500pM, 1aM to 200pM, 1aM to 100pM, 1aM to 10pM, 1aM to 1pM, 100aM to 1nM, 100aM to 500pM, 100aM to 200pM, 100aM to 100pM, 100aM to 10pM, 100aM to 1pM, 250aM to 1nM, 250aM to 500pM, 250aM to 200pM, 250 aM to 100pM, 250aM to 10pM, 250aM to 1pM, 500aM to 1nM, 500aM to 500pM, 500aM to 200pM, 500aM to 100pM, 500aM to 10pM, 500aM to 1pM, 750aM to 1nM, 750aM to 500pM, 750aM to 200pM, 750aM to 100pM, 750aM to 10pM, 750aM to 1pM, 1fM to 1nM, 1fM to 500pM, 1fM to 200pM, 1fM to 100pM, 1fM to 10pM, 1fM to 1pM, 500fM to 500pM, 500fM to 200pM, In some cases, the detection threshold of the method of the present disclosure is in the range of 1aM to 800aM. In some cases, the detection threshold of the method of the present disclosure is in the range of 50aM to 1pM. In some cases, the detection threshold of the method of the present disclosure is in the range of 50aM to 500fM (wherein the concentration refers to the target DNA threshold concentration at which the target DNA can be detected). In some cases, the detection threshold of the method of the present disclosure is in the range of 1aM to 800aM. In some cases, the detection threshold of the method of the present disclosure is in the range of 50aM to 1pM. In some cases, the detection threshold of the method of the present disclosure is in the range of 50aM to 500fM.

[0347] In some cases, the target DNA present in the sample is between 1aM and 1nM (e.g., 1aM to 500pM, 1aM to 200pM, 1aM to 100pM, 1aM to 10pM, 1aM to 1pM, 100aM to 1nM, 100aM to 500pM, 100aM to 200pM, 100aM to 100pM, 100aM to 10pM, 100aM to 1pM, 250aM to 1 nM, 250aM to 500pM, 250aM to 200pM, 250aM to 100pM, 250aM to 10pM, 250aM to 1pM, 500aM to 1nM, 500aM to 500pM, 500aM to 200pM, 500aM to 100pM, 500aM to 10pM, 500aM to 1pM, 750aM to 1nM, 750aM to 500pM, 750aM to 200pM, 750 aM to 100pM, 750aM to 10pM, 750aM to 1pM, 1fM to 1nM, 1 fM to 500pM, 1fM to 200pM, 1fM to 100pM, 1fM to 10pM, 1 fM to 1pM, 500fM to 500pM, 500fM to 200pM, 500fM to 100pM, In some cases, the target DNA present in the sample is in the range of 1aM to 800aM. In some cases, the target DNA present in the sample is in the range of 50aM to 1pM. In some cases, the target DNA present in the sample is in the range of 50aM to 500fM.

[0348] In some cases, the minimum concentration at which the target DNA can be detected in a sample is between 1aM and 1nM (e.g., 1aM to 500pM, 1aM to 200pM, 1aM to 100pM, 1aM to 10pM, 1aM to 1pM, 100aM to 1nM, 100aM to 500pM, 100aM to 200pM, 100aM to 100pM, 100aM to 10pM, 100aM to 1pM, 250aM to 1nM, 250aM to 500pM, 250aM to 200pM, 250aM to 100pM, 250aM to 10pM, 250aM to 1pM, 500aM to 1nM, 500aM to 1nM, to 500pM, 500aM to 200pM, 500aM to 100pM, 500aM to 10pM, 500aM to 1pM, 750aM to 1nM, 750aM to 500pM, 750aM to 200pM, 750aM to 100pM, 750aM to 10pM, 750aM to 1pM, 1fM to 1nM, 1fM to 500pM, 1fM to 200pM, 1fM to 100pM, 1fM to 10pM, 1fM to 1pM, 500fM to 500pM, 500fM to 200pM, 500 fM to 100pM, 500fM to 10pM, 500fM to 1pM, 800fM to 1nM, In some cases, the minimum concentration at which the target DNA can be detected in the sample is in the range of 1aM to 500pM. In some cases, the minimum concentration at which the target DNA can be detected in the sample is in the range of 100aM to 500pM.

[0349] In some cases, the target DNA present in the sample is between 1aM and 1nM (e.g., 1aM to 500pM, 1aM to 200pM, 1aM to 100pM, 1aM to 10pM, 1aM to 1pM, 100aM to 1nM, 100aM to 500pM, 100aM to 200pM, 100aM to 100pM, 100aM to 10pM, 100aM to 1pM, 250aM to 1 nM, 250aM to 500pM, 250aM to 200pM, 250aM to 100pM, 250aM to 10pM, 250aM to 1pM, 500aM to 1nM, 500aM to 500pM, 500aM to 200pM, 500aM to 100pM, 500aM to 10pM, 500aM to 1pM, 750aM to 1nM, 750aM to 500pM, 750aM to 200pM, 750 aM to 100pM, 750aM to 10pM, 750aM to 1pM, 1fM to 1nM, 1 fM to 500pM, 1fM to 200pM, 1fM to 100pM, 1fM to 10pM, 1 fM to 1pM, 500fM to 500pM, 500fM to 200pM, 500fM to 100pM, In some cases, the target DNA present in the sample is in the range of 1aM to 500pM. In some cases, the target DNA present in the sample is in the range of 100aM to 500pM.

[0350] In some cases, the subject compositions or methods exhibit attomole (aM) detection sensitivity. In some cases, the subject compositions or methods exhibit femtomolar (fM) detection sensitivity. In some cases, the subject compositions or methods exhibit picomolar (pM) detection sensitivity. In some cases, the subject compositions or methods exhibit nanomolar (nM) detection sensitivity.

[0351] Target DNA

[0352] The target DNA can be single-stranded (ssDNA) or double-stranded (dsDNA). When the target DNA is single-stranded, there is no preference or requirement for the PAM sequence in the target DNA. However, when the target DNA is dsDNA, the PAM is generally present adjacent to the target sequence of the target DNA (e.g., see discussion of PAM elsewhere herein). The source of the target DNA can be the same as the source of the sample, for example, as described below.

[0353] The source of the target DNA can be any source. In some cases, the target DNA is viral DNA (e.g., genomic DNA of a DNA virus). Thus, the subject method can be used to detect the presence of viral DNA in a nucleic acid population (e.g., in a sample). The subject method can also be used to cut non-target ssDNA in the presence of target DNA. For example, if the method occurs in a cell, then when there is a specific target DNA in the cell, the subject method can be used to promiscuously cut non-target ssDNA (ssDNA that does not hybridize with the guide sequence of the guide RNA) in the cell (e.g., when the cell is infected with a virus and a viral target DNA is detected).

[0354] Examples of possible target DNA include, but are not limited to, viral DNA, such as papovaviruses (e.g., human papillomavirus (HPV), polyomavirus); hepadnaviruses (e.g., hepatitis B virus (HBV)); herpesviruses (e.g., herpes simplex virus (HSV), varicella zoster virus (VZV), Epstein-Barr virus (Epstein-Barr virus); virus, EBV), cytomegalovirus (CMV), herpes lymphotropic virus, rose pityriasis, Kaposi's sarcoma-associated herpesvirus); adenovirus (e.g., avian adenovirus, avian adenovirus, fish adenovirus (ichtadenovirus), American white sturgeon adenovirus (mastadenovirus), sialidase adenovirus); poxvirus (e.g., smallpox, vaccinia virus, cowpox virus, monkeypox virus, aphthous stomatitis virus, pseudovaccinia virus, bovine papular stomatitis virus; Tena poxvirus, Yaba monkey tumor virus; molluscum contagiosum virus (MCV)); parvovirus (e.g., adeno-associated virus (AAV), parvovirus B19, human bocavirus, bufavirus, human parv4G1); Geminiviridae; Dwarf Viridae; Phycodnaviridae, etc. In some cases, the target DNA is parasitic DNA. In some cases, the target DNA is bacterial DNA, such as DNA of pathogenic bacteria.

[0355] sample

[0356] The subject sample includes nucleic acids (e.g., multiple nucleic acids). The term "multiple" is used herein to mean two or more. Thus, in some cases, the sample comprises two or more (e.g., 3 or more, 5 or more, 10 or more, 20 or more, 50 or more, 100 or more, 500 or more, 1,000 or more, or 5,000 or more) nucleic acids (e.g., DNA). The subject method can be used as a very sensitive method for detecting target DNA present in a sample (e.g., in a complex mixture of nucleic acids such as DNA). In some cases, the sample comprises 5 or more DNAs (e.g., 10 or more, 20 or more, 50 or more, 100 or more, 500 or more, 1,000 or more, or 5,000 or more DNAs) whose sequences are different from each other. In some cases, the sample comprises 10 or more, 20 or more, 50 or more, 100 or more, 500 or more, 1 ... 3 or more, 5x 10 3 or more, 10 4 or more, 5x 10 4 or more, 10 5 or more, 5x 10 5 or more, 10 6 or more, 5x 10 6 or more, or 10 7 In some cases, the sample contains 10 to 20, 20 to 50, 50 to 100, 100 to 500, 500 to 10 3 10 3 Up to 5x 10 3 pcs, 5x 10 3 Up to 10 4 10 4 Up to 5x 10 4 pcs, 5x 10 4 Up to 10 5 10 5 Up to 5x 10 5 pcs, 5x 10 5 Up to 10 6 10 6 Up to 5x 10 6 or 5x 10 6 Up to 10 7 or more than 10 7 In some cases, the sample contains 5 to 10 7 DNA (e.g., sequences that differ from each other) (e.g., 5 to 106 5 to 10 5 5 to 50,000, 5 to 30,000, 10 to 10 6 10 to 10 5 10 to 50,000, 10 to 30,000, 20 to 10 6 20 to 10 5 In some cases, the sample comprises DNA from a cell lysate (e.g., eukaryotic cell lysate, mammalian cell lysate, human cell lysate, prokaryotic cell lysate, plant cell lysate, etc.). For example, in some cases, the sample comprises DNA from a cell such as a eukaryotic cell, for example a mammalian cell such as a human cell.

[0357] The term "sample" is used herein to refer to any sample comprising DNA (e.g., to determine whether a target DNA is present in a DNA population). The sample may be derived from any source, for example, the sample may be a synthetic combination of purified DNA; the sample may be a cell lysate, a cell lysate rich in DNA, or DNA separated and / or purified from a cell lysate. The sample may be from a patient (e.g., for diagnostic purposes). The sample may be from permeabilized cells. The sample may be from cross-linked cells. The sample may be in a tissue section. The sample may be from a tissue prepared by cross-linking, followed by defatting and adjusting to form a uniform refractive index. An example of a tissue prepared by cross-linking, followed by defatting and adjusting to form a uniform refractive index is described in, for example, Shah et al., Development (2016) 143, 2862-2867 doi: 10.1242 / dev.138560.

[0358] A "sample" may include a target DNA and a plurality of non-target DNAs. In some cases, the target DNA is present in the sample at a concentration of one copy per 10 non-target DNAs, one copy per 20 non-target DNAs, one copy per 25 non-target DNAs, one copy per 50 non-target DNAs, one copy per 100 non-target DNAs, one copy per 500 non-target DNAs, one copy per 10 3 One copy of non-target DNA, 5x10 3 One copy of non-target DNA, one copy of every 10 4 One copy of non-target DNA, 5x 10 4 One copy of non-target DNA, one copy of every 10 5 One copy of non-target DNA, 5x 10 5 One copy of non-target DNA, one copy of every 10 6One copy of non-target DNA or less than one copy per 10 6 In some cases, the target DNA is present in the sample at a rate of 1 copy per 10 non-target DNAs to 1 copy per 20 non-target DNAs, 1 copy per 20 non-target DNAs to 1 copy per 50 non-target DNAs, 1 copy per 50 non-target DNAs to 1 copy per 100 non-target DNAs, 1 copy per 100 non-target DNAs to 1 copy per 500 non-target DNAs, 1 copy per 500 non-target DNAs to 10 3 1 copy of non-target DNA, 10 3 1 copy of non-target DNA to 5x 10 3 1 copy of non-target DNA, 5x 10 3 1 copy of non-target DNA to 10 4 1 copy of non-target DNA, 10 4 1 copy of non-target DNA to 10 5 1 copy of non-target DNA, 10 5 1 copy of non-target DNA to 10 6 1 copy of non-target DNA, or 10 6 1 copy of non-target DNA to 10 7 One copy of non-target DNA is present.

[0359] Suitable samples include, but are not limited to, saliva, blood, serum, plasma, urine, aspirates, and biopsy samples. Therefore, the term "sample" with respect to a patient covers blood and other liquid samples of biological origin, solid tissue samples such as biopsy specimens or tissue cultures, or cells and their offspring derived therefrom. The definition also includes samples that have been manipulated in any of the following ways after acquisition: such as treatment with reagents; washing; or enrichment for certain cell populations such as cancer cells. The definition also includes samples that have been enriched with molecules of a specific type (e.g., DNA). The term "sample" covers biological samples, such as clinical samples, such as blood, plasma, serum, aspirates, cerebrospinal fluid (CSF), and also includes tissues obtained by surgical resection, tissues obtained by biopsy, cells in culture, cell supernatants, cell lysates, tissue samples, organs, bone marrow, etc. "Biological sample" includes biological fluids (e.g., cancer cells, infected cells, etc.) derived therefrom, such as samples containing DNA obtained from such cells (e.g., cell lysates or other cell extracts containing DNA).

[0360] The sample may contain or may be obtained from any of a variety of cells, tissues, organs or cell-free fluids. Suitable sample sources include eukaryotic cells, bacterial cells and archaeal cells. Suitable sample sources include unicellular organisms and multicellular organisms. Suitable sample sources include unicellular eukaryotic organisms; plants or plant cells; algae cells, such as brown grape algae, Chlamydomonas reinhardtii, marine oil-rich microchlorophyll, spreading branched sargassum, feather algae, etc.; fungal cells (e.g., yeast cells); animal cells, tissues or organs; cells, tissues or organs from invertebrates (e.g., fruit flies, cnidarians, echinoderms, nematodes, insects, arachnids, etc.); cells, tissues, body fluids or organs from vertebrates (e.g., fish, amphibians, reptiles, birds, mammals); cells, tissues, body fluids or organs from mammals (e.g., humans; non-human primates; ungulates; felines; cattle; sheep; goats, etc.). Suitable sample sources include nematodes, protozoa, etc. Suitable sample sources include parasites, such as helminths, malarial parasites, and the like.

[0361] Suitable sample sources include, for example, cells, tissues or organisms from any of the following six kingdoms: Bacteria (e.g., Eubacteria); Archaea; Protista; Fungi; Plantae; and Animalia. Suitable sample sources include plant-like members of the Protista kingdom, including but not limited to algae (e.g., green algae, red algae, gray algae, cyanobacteria); fungal-like members of the Protista kingdom, such as slime molds, water molds, etc.; animal-like members of the Protista kingdom, such as flagellates (e.g., Euglena), amoeboids (e.g., amoeba), sporozoans (e.g., Apicomplexa, Myxozoa, Microsporidia) and ciliates (e.g., Paramecium). Suitable sample sources include members of the kingdom Fungi, including but not limited to members of any of the following phyla: Basidiomycota (basidiomycetes; e.g., Agaricus, Amanita, Boletus, Chanterelles, etc.); Ascomycota (ascomycetes, including, e.g., yeasts); Mycophyta (lichens); Zygomycota (zygomycetes); and Imperfectomycetes. Suitable sample sources include members of the kingdom Plantae, including but not limited to members of any of the following classifications: Bryophyta (e.g., mosses), Hornworts (e.g., hornworts), Liverworts (e.g., liverworts), Lycophyta (e.g., Lycophytes), Sphenopteridophyta (e.g., Equisetum), Gymnophyta (e.g., pine ferns), Pteridophyta, Pteridophyta (e.g., ferns), Cycads, Ginkgophylla, Conifers, Gnetum, and Magnolia (e.g., flowering plants).Suitable sample sources include members of the kingdom Animalia, including, but not limited to, any of the following phyla: Porifera (sponges); Platyhelminthes; Orthomozoa (parasites of marine invertebrates); Rhomboids; Cnidaria (corals, sea anemones, jellyfish, sea pens, sea renillas, cubical jellyfish); Ctenophora (ctenophores); Platyhelminthes (flatworms); Nemerteans (nemerteans); Gnathostomulida (jawed worms); Gastrochaeta; Rotifera; Branchiata; Kinorhynchus; Armorata; Acanthocephala; Entoprocta; Nematoda; Nematodes; Cyclostomata; Mollusca (molluscs); Siegeworms (tegument worms); (peanutworms); Annelida (anneled worms); Tardigrades (slow-walking animals); Claws (combroid silkworms); Arthropoda (including the following subphyla: Chelicerata, Myriapoda, Hexapoda, and Crustacea, wherein Chelicerata includes, for example, Arachnida, Arthralocromyces, and Sea Arachnids, wherein Myriapoda includes, for example, Chilopoda (lippods), Diplopoda (polypods), Paropoda, and Synthetia, wherein Hexapoda includes Insecta, and wherein Crustacea includes shrimps, krill, barnacles, etc.; Broomworms; Exoanaria (bryozoans); Brachiopoda; Echinodermata (e.g., starfish, sea daisy, hair star, sea urchin, sea cucumber, brittle star, brittle basket, etc.); baskets, etc.); Chaetognatha (arrow worms); Hemichordata (jade hookworms); and Chordata. Suitable members of the phylum Chordata include any member of the following subphyla: Uzumaki (class Ascidian; including the orders Ascidiana, Salpae, and Larvae); Cephalochordae (amphix); Hagfishes (hagfish); and Vertebrata, wherein members of the subphylum Vertebrata include, for example, members of the following classes: Gnathoidea (lampregnant fish), Chondrichthyes (cartilaginous fish), Actinopterygii (ray-finned fish), Coelacanths (coelacanths), Lungfishes (lungfish), Reptilia (reptiles, such as snakes, alligators, crocodiles, lizards, etc.), Aves (birds); and Mammalia (mammals). Suitable plants include any monocots and any dicots.

[0362] Suitable sample sources include cells, fluids, tissues or organs taken from an organism; specific cells or cell groups isolated from an organism, etc. For example, when the organism is a plant, suitable sources include xylem, phloem, cambium, leaves, roots, etc. When the organism is an animal, suitable sources include specific tissues (e.g., lung, liver, heart, kidney, brain, spleen, skin, fetal tissue, etc.), or specific cell types (e.g., neuronal cells, epithelial cells, endothelial cells, astrocytes, macrophages, glial cells, pancreatic islet cells, T lymphocytes, B lymphocytes, etc.).

[0363] In some cases, the sample source is a diseased (or suspected diseased) cell, fluid, tissue or organ. In some cases, the sample source is a normal (non-diseased) cell, fluid, tissue or organ. In some cases, the sample source is (or suspected to be) a cell, tissue or organ infected by a pathogen. For example, the sample source can be an individual who may or may not be infected - and the sample can be any biological sample collected from the individual (e.g., blood, saliva, biopsy, plasma, serum, bronchoalveolar lavage fluid, sputum, stool sample, cerebrospinal fluid, fine needle aspirate, swab sample (e.g., cheek swab, cervical swab, nasal swab), interstitial fluid, synovial fluid, nasal secretions, eye Tears, buffy coats, mucosal samples, epithelial cell samples (e.g., epithelial cell scrapings), etc.). In some cases, the sample is a cell-free liquid sample. In some cases, the sample is a liquid sample that may contain cells. Pathogens include viruses, fungi, worms, protozoa, malarial parasites, Plasmodium parasites, Toxoplasma parasites, Schistosoma parasites, etc. "Herms" include roundworms, heartworms, and phytophagous nematodes (Nematoda), flukes (Felata), Acanthocephala, and tapeworms (Cestoda). Protozoan infections include those from Giardia species, Trichomonas species, African trypanosomiasis, amebic dysentery, babesiosis, bagworm dysentery, Chagas disease (Chaga's disease, coccidiosis, malaria, and toxoplasmosis. Examples of pathogens (such as parasitic / protozoan pathogens) include, but are not limited to, Plasmodium falciparum, Plasmodium vivax, Trypanosoma cruzi, and Toxoplasma gondii. Fungal pathogens include, but are not limited to, Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, and Candida albicans. albicans). Pathogenic viruses include, for example, immunodeficiency viruses (e.g., HIV); influenza virus; dengue virus; West Nile virus; herpes virus; yellow fever virus; hepatitis C virus; hepatitis A virus; hepatitis B virus; papillomavirus, etc. Pathogenic viruses may include DNA viruses such as papovaviruses (e.g., human papillomavirus (HPV), polyomavirus); hepadnaviruses (e.g., hepatitis B virus (HBV)); herpesviruses (e.g., herpes simplex virus (HSV), varicella zoster virus(VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpes lymphotropic virus, pityriasis rosea, Kaposi's sarcoma-associated herpesvirus); adenovirus (e.g., avian adenovirus, fowl adenovirus, fish adenovirus, American white sturgeon adenovirus, sialidase adenovirus); poxvirus (e.g., smallpox, vaccinia virus, cowpox virus, monkeypox virus, aphthous stomatitis virus, pseudovaccinia virus, bovine papular stomatitis virus; Tena poxvirus, Yaba monkey tumor virus; molluscum contagiosum virus (MCV)); parvovirus (e.g., adeno-associated virus (AAV), parvovirus B19, human bocavirus, bufavirus, human parv4G1); Geminiviridae; Cynomorviridae; Algae family, etc. Pathogens may include, for example, DNA viruses [e.g., papovaviruses (e.g., human papillomavirus (HPV), polyomavirus); hepatotropic DNA viruses (e.g., hepatitis B virus (HBV)); herpes viruses (e.g., herpes simplex virus (HSV), varicella zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpes lymphotropic virus, pityriasis rosea, Kaposi's sarcoma-associated herpes virus); adenoviruses (e.g., avian adenovirus, fowl adenovirus, fish adenovirus, American adenovirus); poxviruses (e.g., smallpox, vaccinia virus, cowpox virus, monkeypox virus, aphthous stomatitis virus, pseudovaccinia virus, papular stomatitis virus, Tena poxvirus, Yaba monkey tumor virus, molluscum contagiosum virus (MCV)); parvoviruses (e.g., adeno-associated virus (AAV), parvovirus B19, human bocavirus, bufavirus, human parv4G1); Geminiviridae; Cynomorviridae; Algae family, etc.], Mycobacterium tuberculosis (Mycobacterium tuberculosis, Streptococcus agalactiae, methicillin-resistant Staphylococcus aureus, Legionella pneumophila, Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhoeae, Neisseria meningitidis, Pneumococcus, Cryptococcus neoformans, Histoplasma capsulatum, Hemophilus influenzae B, Treponema pallidumpallidum, Lyme disease spirochetes, Pseudomonas aeruginosa, Mycobacterium leprae, Brucella abortus, rabies virus, influenza virus, cytomegalovirus, herpes simplex virus I, herpes simplex virus II, human serum parvo-like virus, respiratory syncytial virus, varicella-zoster virus, hepatitis B virus, hepatitis C virus, measles virus, adenovirus, human T-cell leukemia virus, Epstein-Barr virus, murine leukemia virus, mumps virus, vesicular stomatitis virus, Sindbis virus, lymphocytic choriomeningitis virus, wart virus, blue tongue virus, Sendai virus virus), feline leukemia virus, reovirus, poliovirus, simian virus 40, mouse mammary tumor virus, dengue virus, rubella virus, West Nile virus, Plasmodium falciparum, Plasmodium vivax, Toxoplasma gondii, Trypanosoma rangeli, Trypanosoma cruzi, Trypanosoma roesiense, Trypanosoma brucei, Schistosoma mansoni, Schistosoma japonicum, Babesia bovis, Eimeria tenella, Onchocerca volvulus, Leishmania tropica, Mycobacterium tuberculosis, Trichinella spiralis, Theileria parva, Taenia hydatigena, Taenia ovis, Taenia saginata, Echinococcus granulosus, Mesocestoides corti, Mycoplasma arthritidis, M. hyorhinis, M. orale, M. arginini, Acholeplasma laidlawii, M. salivarium, and M. pneumoniae.

[0364] Measuring detectable signals

[0365] In some cases, the subject method includes a measuring step (e.g., measuring a detectable signal generated by CasZ-mediated ssDNA cleavage). Because the CasZ polypeptide cuts non-targeted ssDNA once activated (the activation occurs when the guide RNA hybridizes to the target DNA in the presence of the CasZ polypeptide (and in some cases also tranc RNA)), the detectable signal can be any signal generated when the ssRNA is cut. For example, in some cases, the measuring step can include one or more of the following: gold nanoparticle-based detection (e.g., see Xu et al., Angew Chem Int Ed Engl. 2007;46(19):3468-70; and Xia et al., Proc Natl Acad Sci USA. 2010 Jun 15;107(24):10837-41), fluorescence polarization, colloidal phase transition / dispersion (e.g., Baksh et al., Nature. 2004 Jan 8;427(6970):139-41), electrochemical detection, semiconductor-based sensing (e.g., Rothberg et al., Nature. 2011 Jul 20;475(7356):348-52; for example, in ssDNA. The cleavage reaction is followed by the use of a phosphatase to produce a pH change by opening the 2'-3' cyclic phosphate and by releasing inorganic phosphate into the solution), and detection of the labeled detector ssDNA (see elsewhere herein for more details). The readout of such detection methods can be any convenient readout. Examples of possible readouts include, but are not limited to: the amount of detectable fluorescent signal measured; visual analysis of bands on a gel (e.g., bands representing cleavage products versus uncleaved substrate), visual or sensor-based detection of the presence or absence of color (i.e., color detection methods), and the presence or absence of an electrical signal (or a specific amount of an electrical signal).

[0366] In some cases, for example, the amount of the detected signal can be used to determine the amount of the target DNA present in the sample, and the measurement can be quantitative. In some cases, for example, the presence or absence of a detectable signal can indicate the presence or absence of a target DNA (e.g., a virus, a SNP, etc.), and the measurement can be qualitative. In some cases, unless the target DNA (e.g., a virus, a SNP, etc.) is present at a concentration higher than a specific threshold, the detectable signal will not exist (e.g., higher than a given threshold level). In some cases, the detection threshold can be titrated by modifying the amount of the CasZ polypeptide, the guide RNA, the sample volume and / or the detection agent ssDNA (if used). Therefore, for example, as will be understood by those of ordinary skill in the art, if necessary, many controls can be used to establish one or more reactions, each reaction being set for detecting different threshold levels of target DNA, and therefore such a series of reactions can be used to determine the amount of the target DNA present in the sample (e.g., such a series of reactions can be used to determine that the target DNA 'is present in the sample at least at a concentration of X'). Non-limiting examples of applications / uses of the compositions and methods of the present disclosure include single nucleotide polymorphism (SNP) detection, cancer screening, bacterial infection detection, antibiotic resistance detection, viral infection detection, etc. The compositions and methods of the present disclosure can be used to detect any DNA target. For example, any virus that integrates nucleic acid material into the genome can be detected because the test sample can contain cell genomic DNA-and the guide RNA can be designed to detect the integrated nucleotide sequence. In some cases, the methods of the present disclosure do not include an amplification step. In some cases, the methods of the present disclosure include an amplification step.

[0367] In some cases, the disclosed methods can be used to determine the amount of target DNA in a sample (e.g., a sample comprising a target DNA and a plurality of non-target DNAs). Determining the amount of target DNA in a sample can include comparing the amount of detectable signal generated by a test sample with the amount of d...

Claims

1. A method for directing a polypeptide to a target sequence of a target nucleic acid, The method comprises contacting the target nucleic acid with an engineered and / or non-naturally occurring complex comprising: (a) a polypeptide, wherein the polypeptide consists of the amino acid sequence of SEQ ID NO: 3; and (b) a guide RNA comprising a guide sequence that hybridizes to a target sequence of the target nucleic acid, wherein the polypeptide binds to a region of the guide RNA to form a complex that interacts with the target nucleic acid, The methods described herein are not methods of treating disease in animals or humans.

2. The method of claim 1, wherein the method results in modification of the target nucleic acid, regulation of transcription from the target nucleic acid, or modification of a polypeptide associated with the target nucleic acid. The method of claim 2 , wherein the target nucleic acid is modified by being cleaved.

4. The method of any one of claims 1 to 3, wherein the target nucleic acid is selected from the group consisting of double-stranded DNA, single-stranded DNA, RNA, genomic DNA, and extrachromosomal DNA.

5. The method of any one of claims 1 to 3, wherein the guide sequence and the region that binds to the polypeptide are heterologous to each other.

6. The method of any one of claims 1-3, wherein the contacting results in genome editing.

7. The method of any one of claims 1-3, wherein the contacting occurs outside a bacterial cell and outside an archaeal cell.

8. The method of any one of claims 1-3, wherein the contacting occurs outside of a cell in vitro.

9. The method of any one of claims 1-3, wherein the contacting occurs inside a target cell.

10. The method of claim 9, wherein the contacting comprises: Introducing into the target cell at least one of the following: (a) the polypeptide or a nucleic acid encoding the polypeptide; and (b) the guide RNA or a nucleic acid encoding the guide RNA.

11. The method of claim 10, wherein the nucleic acid encoding the polypeptide is a non-native sequence that is codon-optimized for expression in the target cell.

12. The method of claim 9, wherein the target cell is a eukaryotic cell.

13. The method of claim 9, wherein the target cells are in in vitro culture.

14. The method of claim 9, wherein the target cell is in vivo.

15. The method of claim 9, wherein the target cell is ex vivo.

16. The method of claim 12, wherein the eukaryotic cell is selected from the group consisting of a plant cell, a fungal cell, a unicellular eukaryotic organism, and an animal cell.

17. The method of claim 9, wherein the contacting further comprises: A DNA donor template is introduced into the target cells.

18. The method of claim 1, wherein the method comprises contacting the target nucleic acid with a trans-activating noncoding RNA (trancRNA).

19. The method of claim 9, wherein the contacting comprises: A trans-activating non-coding RNA (trancRNA) and / or a nucleic acid encoding the trancRNA is introduced into the target cell.

20. The method of claim 18 or claim 19, wherein the nucleic acid sequence of the trancRNA is shown in SEQ ID NO:

153.

21. The method of any one of claims 10-19, wherein at least one of the nucleic acid encoding the polypeptide and the nucleic acid encoding the guide RNA is a recombinant expression vector.

22. The method of any one of claims 18-19, wherein the nucleic acid encoding the trancRNA is a recombinant expression vector.

23. The method of claim 21, wherein the expression vector is an adenovirus-associated vector.

24. The method of any one of claims 10-19, wherein the nucleic acid encoding the polypeptide is mRNA.

25. The method of any one of claims 1-3, wherein the polypeptide is conjugated to a heterologous polypeptide, or the nucleic acid encoding the polypeptide encodes a fusion polypeptide comprising the polypeptide and a heterologous polypeptide.

26. The method of claim 25, wherein the heterologous polypeptide is a transcriptional repressor or a transcriptional activator.

27. The method of claim 25, wherein the heterologous polypeptide is selected from the group consisting of an acetyltransferase, a deacetylase, a methyltransferase, a demethylase, a deaminase, a transposase, an integrase, a polymerase, and a helicase.

28. The method of any one of claims 1-3 and 10-19, wherein the guide RNA comprises one or more of the following: modified nucleobases, modified backbones or non-natural internucleoside linkages, modified sugar moieties, locked nucleic acids, peptide nucleic acids, and deoxyribonucleotides.

29. The method of any one of claims 18-19, wherein the trancRNA comprises one or more of the following: modified nucleobases, modified backbones or non-natural internucleoside linkages, modified sugar moieties, locked nucleic acids, peptide nucleic acids, and deoxyribonucleotides.

30. The method of claim 12, wherein the eukaryotic cell is selected from the group consisting of an invertebrate cell and a vertebrate cell.

31. The method of claim 12, wherein the eukaryotic cell is selected from the group consisting of a mammalian cell, a reptile cell, an insect cell, an avian cell, a fish cell, a parasite cell, and an arthropod cell.

32. The method of claim 12, wherein the eukaryotic cell is selected from the group consisting of a rodent cell and a primate cell.

33. The method of claim 12, wherein the eukaryotic cell is selected from the group consisting of a mouse cell, a rat cell, a non-human primate cell, and a human cell.

34. A composition comprising an engineered and / or non-naturally occurring complex comprising: (a) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 3, or a nucleic acid encoding the polypeptide; and (b) a guide RNA or a nucleic acid encoding the guide RNA, wherein the guide RNA comprises a guide sequence complementary to a target sequence of a target nucleic acid and comprises a region that binds to the polypeptide.

35. The composition of claim 34, further comprising a trans-activated non-coding RNA (trancRNA) or a nucleic acid encoding the trancRNA.

36. The composition of claim 34, comprising lipid nanoparticles.

37. A kit comprising an engineered and / or non-naturally occurring complex comprising: (a) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 3, or a nucleic acid encoding the polypeptide; (b) a guide RNA or a nucleic acid encoding the guide RNA, wherein the guide RNA comprises a guide sequence complementary to a target sequence of a target nucleic acid and comprises a region that binds to the polypeptide.

38. The kit of claim 37, further comprising a trans-activated non-coding RNA (trancRNA) or a nucleic acid encoding the trancRNA.

39. A genetically modified eukaryotic cell comprising at least one of the following: (a) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 3, or a nucleic acid encoding the polypeptide; (b) a guide RNA or a nucleic acid encoding the guide RNA, wherein the guide RNA comprises a guide sequence complementary to a target sequence of a target nucleic acid and comprises a region that binds to the polypeptide; and (c) a trans-activated non-coding RNA (trancRNA) or a nucleic acid encoding said trancRNA, The eukaryotic cell is an in vitro cell and is not a plant cell or a reproductive cell.

40. The composition of any one of claims 34-36, the kit of any one of claims 37-38, or the eukaryotic cell of claim 39, characterized in that At least one of the following: (a) the nucleic acid encoding the polypeptide comprises a nucleotide sequence that: (i) encodes the polypeptide, and (ii) is operably linked to a heterologous promoter; (b) the nucleic acid encoding the guide RNA comprises a nucleotide sequence that: (i) encodes the guide RNA, and (ii) is operably linked to a heterologous promoter; and (c) the nucleic acid encoding the trancRNA comprises a nucleotide sequence that: (i) encodes the trancRNA, and (ii) is operably linked to a heterologous promoter.

41. The composition of any one of claims 34-36, the kit of any one of claims 37-38, or the eukaryotic cell of claim 39, wherein at least one of the following is a recombinant expression vector: the nucleic acid encoding the polypeptide, the nucleic acid encoding the guide RNA, and the nucleic acid encoding the trancRNA.

42. The composition of any one of claims 34-36, the kit of any one of claims 37-38, or the eukaryotic cell of claim 39, wherein the guide RNA and / or the trancRNA comprises one or more of the following: a modified nucleobase, a modified backbone or non-natural internucleoside linkage, a modified sugar moiety, a locked nucleic acid, a peptide nucleic acid, and a deoxyribonucleotide.

43. The composition of any one of claims 34-36, the kit of any one of claims 37-38, or the eukaryotic cell of claim 39, wherein at least one of the following is conjugated to a heterologous moiety: the polypeptide, the nucleic acid encoding the polypeptide, the guide RNA, the nucleic acid encoding the guide RNA, the trancRNA, and the nucleic acid encoding the trancRNA.

44. The composition, kit or eukaryotic cell of claim 43, wherein the heterologous moiety is a heterologous polypeptide.

45. The composition, kit or eukaryotic cell of claim 44, wherein the heterologous polypeptide: (i) has DNA modification activity, (ii) exhibits the ability to increase or decrease transcription, and / or (iii) has enzymatic activity that modifies a polypeptide associated with DNA.

46. ​​The composition of any one of claims 34-36, the kit of any one of claims 37-38, or the eukaryotic cell of claim 39, wherein the guide sequence and the region that binds to the polypeptide are heterologous to each other.

47. A method for detecting a target DNA in a sample, the method comprising: (a) contacting the sample with: (i) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 3; (ii) a guide RNA comprising: a region that binds to the polypeptide and a guide sequence that hybridizes to the target DNA; and (iii) a detector DNA that is single-stranded and does not hybridize to the guide sequence of the guide RNA; as well as (b) measuring a detectable signal generated by cleavage of the single-stranded detector DNA by the polypeptide, thereby detecting the target DNA.

48. The method of claim 47, wherein the target DNA is single-stranded.

49. The method of claim 47, wherein the target DNA is double-stranded.

50. The method of any one of claims 47-49, wherein the target DNA is viral DNA.

51. The method of any one of claims 47-49, wherein the target DNA is papovavirus, hepadnavirus, herpesvirus, adenovirus, poxvirus, or parvovirus DNA.

52. The method of any one of claims 47-49, wherein the sample comprises DNA molecules from cell lysate.

53. The method of any one of claims 47-49, wherein the sample comprises cells.

54. The method of any one of claims 47-49, wherein the contacting is performed inside a cell in vitro, ex vivo, or in vivo.

55. The method of claim 54, wherein the cell is a eukaryotic cell.

56. The method of any one of claims 47-49, wherein the target DNA can be detected at a concentration as low as 10 aM.

57. The method of any one of claims 47-49, comprising determining the amount of the target DNA present in the sample.

58. The method of claim 57, wherein the determining comprises: measuring the detectable signal to generate a test measurement; measuring a detectable signal produced by a reference sample or cell to generate a reference measurement; as well as The test measurement is compared to the reference measurement to determine the amount of target DNA present in the sample.

59. The method of any one of claims 47-49, wherein measuring the detectable signal comprises one or more of: gold nanoparticle-based detection, fluorescence polarization, colloidal phase transition / dispersion, electrochemical detection, and semiconductor-based sensing.

60. The method of any one of claims 47-49, wherein the single-stranded detector DNA comprises a fluorescent emitting dye pair.

61. The method of claim 60, wherein the fluorescent emitting dye pair produces a detectable signal before the single-stranded detector DNA is cleaved, and the amount of the detectable signal decreases after the single-stranded detector DNA is cleaved.

62. The method of claim 60, wherein the single-stranded detector DNA produces a first detectable signal before being cleaved and produces a second detectable signal after the single-stranded detector DNA is cleaved.

63. The method of any one of claims 61-62, wherein the fluorescent emitting dye pair is a fluorescence resonance energy transfer pair.

64. The method of claim 47, wherein the amount of detectable signal increases after the single-stranded detector DNA is cleaved.

65. The method of claim 60, wherein the fluorescent emitting dye pair is a quencher / fluorescer pair.

66. The method of any one of claims 47, 61-62, and 64-65, wherein the single-stranded detector DNA comprises two or more fluorescent emitting dye pairs.

67. The method of claim 66, wherein the two or more fluorescent emitting dye pairs comprise a fluorescence resonance energy transfer (FRET) pair and a quencher / fluorescer pair.

68. The method of any one of claims 47-49, wherein the single-stranded detector DNA comprises a modified nucleobase, a modified sugar moiety, and / or a modified nucleic acid linkage.

69. The method of any one of claims 47-49, wherein the method comprises amplifying nucleic acids in the sample.

70. The method of claim 69, wherein the amplification comprises isothermal amplification.

71. The method of claim 70, wherein the isothermal amplification comprises recombinase polymerase amplification.

72. The method of claim 69, wherein the amplification begins prior to the contacting of step (a).

73. The method of claim 69, wherein the amplification is initiated together with the contacting of step (a).

74. A kit for detecting a target DNA in a sample, the kit comprising: (a) a guide RNA or a nucleic acid encoding the guide RNA; wherein the guide RNA comprises: a region that binds to a polypeptide and a guide sequence that is complementary to a target DNA; (b) a labeled detector DNA that is single-stranded and does not hybridize to the guide sequence of the guide RNA; and (c) a polypeptide consisting of the amino acid sequence of SEQ ID NO:

3.

75. The kit of claim 74, wherein the labeled detector DNA comprises a fluorescent emitting dye pair.

76. The kit of claim 75, wherein the fluorescent emitting dye pair is a FRET pair.

77. The kit of claim 75, wherein the fluorescent emitting dye pair is a quencher / fluorescer pair.

78. The kit of any one of claims 75-77, wherein the single-stranded detector DNA comprises two or more fluorescent emitting dye pairs.

79. The kit of claim 78, wherein the two or more fluorescent emitting dye pairs include a first fluorescent emitting dye pair that produces a first detectable signal and a second fluorescent emitting dye pair that produces a second detectable signal.

80. The kit of any one of claims 74-77, further comprising a nucleic acid amplification component.

81. The kit of claim 80, wherein the nucleic acid amplification component is a component for recombinase polymerase amplification.

82. A method for cleaving single-stranded DNA (ssDNA), the method comprising: contacting a population of nucleic acids, wherein the population comprises a target DNA and a plurality of non-target ssDNAs, with: (i) polypeptides; and (ii) a guide RNA, the guide RNA comprising: a region that binds to the polypeptide and a guide sequence that hybridizes with the target DNA, wherein the polypeptide cleaves the plurality of non-target ssDNAs, wherein the polypeptide consists of the amino acid sequence of SEQ ID NO: 3, and The methods described herein are not methods of treating disease in animals or humans.

83. The method of claim 82, wherein the contacting is inside a cell in vitro, ex vivo, or in vivo.

84. The method of claim 83, wherein the cell is a eukaryotic cell.

85. The method of claim 84, wherein the eukaryotic cell is a plant cell.

86. The method of any one of claims 83-85, wherein the non-target ssDNA is exogenous to the cell.

87. The method of claim 86, wherein the non-target ssDNA is viral DNA.

88. The method of any one of claims 82-85, wherein the target DNA is single-stranded.

89. The method of any one of claims 82-85, wherein the target DNA is double-stranded.

90. The method of any one of claims 82-85, wherein the target DNA is viral DNA.

91. The method of any one of claims 82-85, wherein the target DNA is papovavirus, hepadnavirus, herpesvirus, adenovirus, poxvirus, or parvovirus DNA.

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