Crispr-cas system and use thereof

Abstract

Provided are a CRISPR-Cas protein, a composition thereof and a use thereof. The composition comprises the protein or a fusion protein, or a nucleic acid molecule encoding same. Also provided is a method for nucleic acid editing using the protein or fusion protein.

Description

CRISPR-Cas systems and uses thereof

[0001] Cross-reference to Related Applications

[0002] This application claims the benefit of and priority to Chinese Patent Application No. 202411297378.2, filed September 14, 2024, entitled “Polypeptides for gene editing and uses thereof,” including any sequence listing and drawings, which is incorporated by reference herein in its entirety.

[0003] Reference to Electronic Sequence Listing

[0004] The present disclosure contains an electronic sequence listing (which was created using the software “WIPO Sequence” in accordance with WIPO Standard ST.26) which is incorporated by reference herein in its entirety. In accordance with WIPO Standard ST.26, the symbol “t” is used to represent both T in DNA and U in RNA. Thus, in a sequence listing prepared according to ST.26, in any instance in which the sequence is RNA, each T in the sequence should be considered to be a U. TECHNICAL FIELD

[0005] The present disclosure relates generally to systems, methods, and compositions for controlling gene expression involving sequence targeting, such as nucleic acid editing, which can use vector systems related to Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) and components thereof. BACKGROUND

[0006] Clustered regularly interspaced short palindromic repeats (CRISPR) system is formed by bacteria and archaea to defend against invading phage DNA. The immune interference process of CRISPR system mainly includes three stages: adaptation, expression, and interference. In the adaptation stage, the CRISPR system integrates a short fragment of DNA from the phage or plasmid between the leader sequence and the first repeat sequence, and each integration is accompanied by the replication of the repeat sequence, thereby forming a new repeat-spacer sequence unit. In the expression stage, the CRISPR locus is transcribed into a CRISPR RNA (crRNA) precursor (pre-crRNA), which is further processed into small crRNAs at the repeat sequences in the presence of Cas proteins and tracrRNA. The mature crRNA forms a Cas / crRNA complex with Cas proteins. In the interference stage, the crRNA guides the Cas / crRNA complex to find the target through the region complementary to the target sequence, and causes a double-stranded DNA break at the target position through the nuclease activity of the Cas protein, thereby making the target DNA lose its original function.

[0007] CRISPR system is divided into three families of I, II, III type, among which the most common type II system is CRISPR / Cas9 system, Cas9 protein can be processed into mature crRNA combined with tracrRNA with the help of trans-encoded small RNA (tracrRNA). After that, it is found that the single-stranded chimeric guide RNA (guide RNA) which simulates the crRNA:tracrRNA complex can effectively mediate the recognition and cutting of Cas9 protein to the target. Among them, the three bases next to the 3' end of the target must be in the form of 5'-NGG-3', thereby forming the PAM (protospacer adjacent motif) structure required for Cas / crRNA complex to recognize the target. However, the different CRISPR / Cas systems currently exist each have different advantages and defects. For example, Cas9, C2c1 and CasX all need two RNA guide RNAs. Common Cas9, C2c1, CasY and Cpf1 are usually about 1300 amino acids in size. In addition, the PAM sequences of Cas9, Cpf1, CasX and CasY are complex and diverse. There is still a need to develop new Cas proteins and CRISPR-Cas systems to meet the diverse application needs.

[0008] The present disclosure provides solutions to these and other needs in the art, and the citation or identification of any document in this application is not an admission that such document is available as prior art to the present disclosure. SUMMARY

[0009] In light of the above background, the present disclosure provides certain advantages and improvements over the prior art.

[0010] While the present disclosure is not limited to particular advantages or functions, in one aspect, the present disclosure provides a Cas protein selected from the group consisting of:

[0011] (a) a polypeptide having an amino acid sequence as set forth in any one of SEQ ID NOs: 1-4;

[0012] (b) a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% homology (or identity) to an amino acid sequence as set forth in any one of SEQ ID NOs: 1-4, and the polypeptide has a biological function as set forth in any one of SEQ ID NOs: 1-4;

[0013] (c) a derivative polypeptide formed by substitution, deletion, or addition of one or more amino acid residues of the amino acid sequence set forth in any one of SEQ ID NOs: 1-4, and retaining the biological function of any one of SEQ ID NOs: 1-4.

[0014] In yet another aspect, the present disclosure provides a fusion protein comprising a Cas protein of the present disclosure and one or more functional domains.

[0015] In yet another aspect, the present disclosure provides a polynucleotide, which is a nucleotide sequence encoding a Cas protein of the present disclosure or a fusion protein of the present disclosure, or a nucleotide sequence encoding a fusion protein of the present disclosure; in some embodiments, the polynucleotide is a DNA molecule codon-optimized according to the codon bias of a host cell; in some embodiments, the host cell comprises a prokaryotic cell or a eukaryotic cell.

[0016] In one embodiment, the DNA molecule comprises a nucleotide having 70% or more (optionally, 90% or more, optionally, 95% or more, optionally, 99% or more, optionally, 100%) identity to the nucleotide sequence set forth in any one of SEQ ID NOs: 5-8.

[0017] In yet another aspect, the present disclosure provides a CRISPR-Cas composition, which comprises:

[0018] (1) a Cas protein of the present disclosure or a fusion protein of the present disclosure, or a nucleic acid molecule encoding a Cas protein of the present disclosure or a fusion protein of the present disclosure;

[0019] (2) a guide RNA, or one or more DNA molecules encoding the guide RNA.

[0020] In yet another aspect, the present disclosure provides a delivery system comprising a Cas protein of the present disclosure, or a fusion protein of the present disclosure, or a polynucleotide of the present disclosure, or a CRISPR-Cas composition of the present disclosure.

[0021] In yet another aspect, the present disclosure provides a host cell or progeny thereof, which comprises a Cas protein of the present disclosure, or a fusion protein of the present disclosure, or a polynucleotide of the present disclosure, or a CRISPR-Cas composition of the present disclosure, or a delivery system of the present disclosure.

[0022] In yet another aspect, the present disclosure provides a method of targeting and editing a target nucleic acid, which comprises contacting the target nucleic acid with a Cas protein of the present disclosure, or a fusion protein of the present disclosure, or a polynucleotide of the present disclosure, or a CRISPR-Cas composition of the present disclosure, or a host cell of the present disclosure.

[0023] In yet another aspect, the present disclosure provides a method of non-specifically degrading single-stranded nucleic acid after recognizing a target nucleic acid, the method comprising contacting a target sequence of the target nucleic acid with the CRISPR-Cas composition of the present disclosure.

[0024] In yet another aspect, the present disclosure provides a method of targeting and cleaving double-stranded target nucleic acid, the method comprising contacting the double-stranded target nucleic acid with the CRISPR-Cas composition of the present disclosure.

[0025] In yet another aspect, the present disclosure provides a method of inducing a change in cell state, the method comprising contacting the CRISPR-Cas composition of the present disclosure with the target nucleic acid in a cell.

[0026] In yet another aspect, the present disclosure provides a kit comprising the Cas protein of the present disclosure, the fusion protein of the present disclosure, or the polynucleotide of the present disclosure, or the CRISPR-Cas composition of the present disclosure, or the host cell of the present disclosure, the components of the kit being in the same or different containers.

[0027] In yet another aspect, the present disclosure provides a method of detecting a target nucleic acid in a sample, the method comprising contacting the sample with the CRISPR-Cas composition of the present disclosure and a non-target sequence; detecting a detectable signal generated by cleavage of the non-target sequence, thereby detecting the target nucleic acid; the non-target sequence does not hybridize to the guide RNA.

[0028] In yet another aspect, the present disclosure provides the Cas protein of the present disclosure, or the fusion protein of the present disclosure, or the polynucleotide of the present disclosure, or the CRISPR-Cas composition of the present disclosure, or the delivery system of the present disclosure, or the host cell of the present disclosure, or the method of targeting and editing a target nucleic acid of the present disclosure, or the method of non-specifically degrading or cleaving single-stranded nucleic acid after recognizing a target nucleic acid of the present disclosure, or the method of targeting and cleaving double-stranded target nucleic acid of the present disclosure in nucleic acid detection.

[0029] In yet another aspect, the present disclosure provides a pharmaceutical composition comprising the CRISPR-Cas composition of the present disclosure, the delivery system of the present disclosure, the host cell of the present disclosure; and a pharmaceutically acceptable excipient;

[0030] In yet another aspect, the present disclosure provides a method for diagnosing, preventing or treating a disorder or disease in a subject in need thereof, the method comprising administering to the subject an effective amount of the Cas protein of the present disclosure, or the fusion protein of the present disclosure, or the polynucleotide of the present disclosure, or the CRISPR-Cas composition of the present disclosure, or the delivery system of the present disclosure, or the host cell of the present disclosure.

[0031] The details of one or more implementations of the present disclosure are set forth in the description below. Other features or advantages of the present disclosure will be apparent from the following drawings and detailed description of several implementations and from the claims. It should be understood that any of the features, concepts, or implementations described herein can be combined with any of the other features, concepts, or implementations described herein, unless specifically noted otherwise. All such modifications and variations are within the scope of the disclosure. BRIEF DESCRIPTION OF DRAWINGS

[0032] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the present disclosure are utilized, and the accompanying drawings of which:

[0033] Figure 1 shows a T7-Cas-T7-crRNA vector map.

[0034] Figure 2 shows secondary structure prediction of direct repeat (DR) sequences corresponding to YTGE-167, YTGE-174, YTGE-190 and YTGE-192.

[0035] Figure 3 shows a Target plasmid map

[0036] Figure 4 shows editing efficiency of YTGE-167, YTGE-174, YTGE-190 and YTGE-192 in E. coli.

[0037] Figure 5 shows cleavage efficiency of YTGE-167, YTGE-174, YTGE-190 and YTGE-192 in mammalian cells.

[0038] The accompanying drawings are for illustrative purposes only and are not necessarily drawn to scale. DETAILED DESCRIPTION

[0039] The following examples are merely illustrative of the present disclosure and do not limit the scope of the disclosure. Unless otherwise expressly stated, experiments and methods described in the examples were performed according to conventional methods well known in the art and as described in various references.

[0040] In addition, unless otherwise indicated, conventional methods and apparatuses known and available to those skilled in the art were used in the examples. Unless otherwise indicated, the reagents and apparatuses used in the examples were obtained from commercial vendors and are conventional products available to those skilled in the art. Those skilled in the art know that the examples describe the present disclosure by way of example only and are not intended to limit the scope of the disclosure as claimed. All publications and other references mentioned herein are incorporated by reference in their entirety.

[0041] SUMMARY

[0042] In this study, the applicants demonstrated that the CRISPR-Cas system provided herein is capable of universal and efficient gene editing in mammalian cells. The applicants discovered multiple Cas proteins, YTGE-167, YTGE-174, YTGE-190 and YTGE-192, which show high editing activity at 5’-TTN-PAM sites.

[0043] The CRISPR-Cas system of the present disclosure has high editing activity, can be delivered in multiple delivery modes, such as AAV or LNP, given its small size, short guide RNA (crRNA), suitable for in vivo multiplex gene editing applications, with great potential for therapeutic genome editing applications.

[0044] In summary, the CRISPR-Cas system described herein has strong editing activity and high specificity, is a universal platform for genome editing or base editing in mammalian cells, and can be used in future in vivo or ex vivo therapeutic applications.

[0045] Cas protein

[0046] As used herein, Cas protein, Cas enzyme, Cas effector protein can be used interchangeably, Cas protein takes its broadest meaning, including wild-type Cas protein, its derivative or variant, analog, and functional fragment thereof such as oligonucleotide binding fragment.

[0047] The term "wild-type" has the meaning generally understood by those skilled in the art, which indicates the typical form of a biological, strain, gene, protein, or a characteristic that distinguishes it from the mutant or variant form when it exists in nature, which can be isolated from sources in nature and has not been intentionally modified by humans.

[0048] In some embodiments, the Cas proteins of the present disclosure are referred to as YTGE-167, YTGE-174, YTGE-190 and YTGE-192, the amino acid sequences of which are shown in SEQ ID NO: 1-4.

[0049] As used herein, the terms "variant", "derivative" and "analog" refer to a polypeptide that substantially maintains the function or activity of the Cas protein of the present disclosure.

[0050] In some embodiments, the Cas protein comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical to the amino acid sequence shown in any one of SEQ ID NO: 1-4.

[0051] In some embodiments, the Cas protein has one or more substitutions, deletions, or additions of amino acids compared to the amino acid sequence of any one of SEQ ID NOs: 1-4, and substantially retains the biological function of the sequence from which it is derived; the one or more substitutions, deletions, or additions of amino acids comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) substitutions, deletions, or additions of amino acids.

[0052] In some embodiments, the Cas protein of the present disclosure is derived from the same species or different species as YTGE-167, YTGE-174, YTGE-190, and YTGE-192.

[0053] Generally, derivatization of a protein does not adversely affect the desired activity of the protein (e.g., activity of binding to a guide RNA, endonuclease activity, activity of binding to and cleaving a target sequence at a specific site under the guidance of a guide RNA), that is, the derivative of the protein has the same activity as the protein. The modified forms of “derivative” include one or more amino acids of the protein can be deleted, inserted, modified, and / or substituted. The terms “non-naturally occurring” or “engineered” are used interchangeably and indicate the involvement of man-made intervention.

[0054] It is clear to one skilled in the art that the structure of a protein can be altered without adversely affecting its activity and functionality, for example, by introducing one or more conservative amino acid substitutions in the amino acid sequence of the protein without adversely affecting the activity and / or three-dimensional structure of the protein molecule.

[0055] Examples of conservative amino acid substitutions and embodiments are clear to one skilled in the art. Specifically, an amino acid residue can be replaced with another amino acid residue belonging to the same group, i.e., a nonpolar amino acid residue is replaced with another nonpolar amino acid residue, a polar uncharged amino acid residue is replaced with another polar uncharged amino acid residue, a basic amino acid residue is replaced with another basic amino acid residue, and an acidic amino acid residue is replaced with another acidic amino acid residue. Such substituted amino acid residues can or can not be encoded by the genetic code. A conservative substitution in which one amino acid is replaced with another amino acid belonging to the same group falls within the scope of the present disclosure as long as the substitution does not result in inactivation of the biological activity of the protein. Thus, the proteins of the present disclosure can comprise one or more conservative substitutions in the amino acid sequence, which are preferably replaced according to Table 1 to produce. In addition, the present disclosure also encompasses proteins comprising one or more other non-conservative substitutions as long as the non-conservative substitution does not significantly affect the desired functions and biological activities of the proteins of the present disclosure.

[0056] Conservative amino acid substitutions can be made at one or more predicted nonessential amino acid residues. A "nonessential" amino acid residue is a residue that can be altered (deleted, substituted, or replaced) without altering the biological activity of the protein, whereas an "essential" amino acid residue is required for biological activity. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Amino acid substitutions can be made in non-conserved regions of the Cas enzyme. In general, such substitutions are not made in conserved amino acid residues, or in amino acid residues that are within a conserved motif, where such residues are required for protein activity. However, it will be appreciated by one skilled in the art that functional variants can have fewer conserved or non-conserved alterations in conserved regions.

[0057] In certain embodiments, selected groups of amino acids that are considered to be conservative substitutions for one another:

[0058] Table 1

[0059] It is known to those skilled in the art that one or more amino acid residues can be altered (substituted, deleted, truncated, or inserted) from the N- and / or C-terminus of a protein while still retaining its functional activity. Thus, proteins having one or more amino acid residues altered from the N- and / or C-terminus of a Cas protein of the disclosure while retaining its desired functional activity are within the scope of the disclosure. These alterations can include alterations introduced by modern molecular methods, such as PCR, including PCR amplification of a protein-encoding sequence by inclusion of an amino acid-encoding sequence in the oligonucleotides used in the PCR amplification.

[0060] It will be recognized that proteins can be altered in various ways including amino acid substitutions, deletions, truncations and insertions, methods for which are generally known to those skilled in the art. For example, amino acid sequence variants of the Cas proteins can be prepared by mutations in the DNA. They can also be prepared by other forms of mutagenesis and / or by directed evolution, for example, using known mutagenic, recombination and / or shuffling methods in combination with appropriate screening methods to identify one or more amino acid substitutions; or one to multiple amino acid deletions and / or one to multiple amino acid insertions.

[0061] It will be recognized by one skilled in the art that these minor amino acid changes in the Cas proteins of the disclosure can occur (e.g., naturally-occurring mutations) or be produced (e.g., using r-DNA technology) without loss of protein function or activity. If the mutations occur in the catalytic domain, active site, or other functional domain of the protein, the properties of the polypeptide can change, but the polypeptide can retain its activity. If the mutations are not close to the catalytic domain, active site, or other functional domain, less impact can be expected.

[0062] A person skilled in the art can identify essential amino acids of a Cas protein according to methods known in the art, e.g. site-directed mutagenesis or protein evolution or bioinformatic analysis. The catalytic domain, active site or other functional domains of a protein can also be determined by physical analysis of the structure, e.g. by techniques such as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling in combination with mutation of putative key site amino acids.

[0063] Orthologue

[0064] As used herein, the term "orthologue" has the meaning generally understood by a person skilled in the art. As a further guidance, an "orthologue" of a protein as described herein refers to a protein belonging to a different species which performs the same or a similar function as the protein it is an orthologue of.

[0065] Nucleic acid cleavage of the present disclosure includes: DNA or RNA break in a target nucleic acid produced by the Cas protein (Cis cleavage), DNA or RNA break in a side branch nucleic acid substrate (single stranded nucleic acid substrate) using Cas protein collateral activity (i.e. non-specific or non-targeted, Trans cleavage). In some embodiments, the cleavage is a double stranded DNA break. In some embodiments, cleavage is a single stranded DNA break or a single stranded RNA break.

[0066] Trans cleavage refers to the fact that in certain circumstances, activated Cas12 family proteins remain active after binding to a target sequence and continue to non-specifically cleave non-target oligonucleotides. This collateral activity can be used to detect the presence of a specific target oligonucleotide using the Cas system. For example, Cas12i systems were engineered to non-specifically cleave ssDNA or transcripts. The collateral activity was used in a high-sensitivity and specific nucleic acid detection platform called SHERLOCK, which can be used for many clinical diagnostics (Gootenberg, J.S. et al. Nucleic acid detection with CRISPR-Cas13a / C2c2. Science 356, 438-442 (2017)).

[0067] CRISPR system

[0068] As used herein, the term "Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-CRISPR-associated (Cas) (CRISPR-Cas) system" or "CRISPR system" are used interchangeably and have the meaning generally understood by those skilled in the art, which generally includes a transcription product or other element related to the expression of a CRISPR-associated ("Cas") gene, or a transcription product or other element capable of directing the activity of the Cas gene.

[0069] In one aspect, the present disclosure also provides a CRISPR-Cas composition, comprising:

[0070] (1) a protein component: a Cas protein of the present disclosure, or a fusion protein of the present disclosure; or a nucleic acid molecule encoding a Cas protein of the present disclosure or a fusion protein of the present disclosure;

[0071] (2) an RNA component: a guide RNA, or one or more nucleic acids encoding the guide RNA, or a precursor RNA of a guide RNA, or a nucleic acid encoding a precursor RNA of a guide RNA; the protein component and the nucleic acid component are associated with each other to form a complex.

[0072] In one embodiment, the CRISPR-Cas composition or system comprises one or more vectors, the one or more vectors comprising:

[0073] (1) a first regulatory element operably linked to a nucleotide sequence encoding the Cas protein or a nucleotide sequence encoding the fusion protein; and

[0074] (2) a second regulatory element operably linked to a nucleotide sequence encoding the guide RNA, the guide RNA comprising:

[0075] (a) a spacer sequence capable of hybridizing to a target sequence of a target nucleic acid; and

[0076] (b) a Direct Repeat (DR) sequence linked to the spacer sequence, capable of directing the Cas protein to bind to the guide RNA to form a CRISPR-Cas complex targeting the target sequence.

[0077] wherein the first regulatory element and the second regulatory element are located on the same or different vectors of the CRISPR-Cas vector system.

[0078] In one embodiment, the first regulatory element or the second regulatory element comprises a promoter, the promoter comprising one or more of an inducible promoter, a constitutive promoter, or a tissue-specific promoter.

[0079] In one embodiment, the promoter comprises one or more of T7, SP6, T3, CMV, EF1a, SV40, PGK1, human beta-actin, CAG, U6, H1, T7, T7lac, araBAD, trp, lac, or Ptac.

[0080] In one embodiment, the first regulatory element and the second regulatory element are located on the same or different vectors.

[0081] In one embodiment, the vector comprises a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, a herpes simplex viral vector, or a phagemid vector.

[0082] In one embodiment, the vector comprises a plasmid vector.

[0083] In one embodiment, the target nucleic acid comprises DNA derived from a eukaryote or DNA derived from a prokaryote.

[0084] In one embodiment, the eukaryote comprises an animal or a plant.

[0085] In one embodiment, the target nucleic acid comprises non-human mammalian DNA, human DNA, insect DNA, avian DNA, reptilian DNA, amphibian DNA, rodent DNA, fish DNA, worm DNA, nematode DNA, or yeast DNA.

[0086] In one embodiment, the non-human mammalian DNA comprises non-human primate DNA.

[0087] In yet another aspect, the present disclosure provides a CRISPR / Cas complex. As used herein, the term "CRISPR / Cas complex" refers to a complex formed by the binding of a gRNA (guide RNA) or a mature crRNA (or guide RNA) to a Cas protein, which comprises a spacer sequence that hybridizes to a target sequence and a DR sequence that binds to a Cas protein, the complex being capable of recognizing and cleaving a target nucleotide that can hybridize to the guide RNA or the mature crRNA.

[0088] In some embodiments, the composition is an activated CRISPR complex, the activated CRISPR complex further comprising: a target sequence of a target nucleic acid that binds to the guide RNA.

[0089] guide RNA (gRNA)

[0090] As used herein, the terms “guide RNA (gRNA),” “mature crRNA,” “guide sequence,” “guide RNA,” or “crRNA” are used interchangeably and have the meaning generally understood by one of skill in the art. Generally, a guide RNA can comprise, or essentially consist of, or consist of a direct repeat (DR) sequence and a spacer sequence.

[0091] In certain instances, a guide sequence (used interchangeably with spacer sequence in the present disclosure) is any polynucleotide sequence that has sufficient complementarity to a target sequence to hybridize to the target sequence and direct specific binding of a CRISPR-Cas complex to the target sequence. In one embodiment, the degree of complementarity between a guide sequence and its corresponding target sequence, when optimally aligned, is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%.

[0092] The crRNA of YTGE-167, YTGE-174, YTGE-190 and YTGE-192 of the present disclosure comprises a spacer sequence that hybridizes to a target nucleic acid, wherein the target nucleic acid comprises a sequence located at the 3’ end of a protospacer adjacent motif (PAM);

[0093] In some embodiments, the PAM sequence is 5’-TTN-3’, wherein N is selected from A, T, C or G.

[0094] In some embodiments, the 3’ end of the Direct Repeat (DR) sequence comprises a stem loop structure, further comprising a stem formed by a first stem nucleotide strand and a second stem nucleotide strand hybridizing to each other to form the stem of the stem loop structure, and a loop formed by the loop nucleotide strand forming the loop of the stem loop structure.

[0095] In some embodiments, the Direct Repeat (DR) sequence comprises one stem loop structure at the 5’ end and one stem loop structure at the 3’ end.

[0096] In some embodiments, the stem of the stem loop structure does not require precise base pairing. Thus, the stem can comprise one or more base mismatches. Alternatively, the base pairing can be precise, i.e. does not comprise any mismatches.

[0097] In some embodiments, the stem of the stem loop structure is separated into two stem portions by a loop.

[0098] In one embodiment, the Direct Repeat (DR) sequence comprises a nucleotide sequence having at least 80% identity to the nucleotide sequence set forth in any one of SEQ ID NOs: 9-12;

[0099] In one embodiment, the Direct Repeat (DR) sequence comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% identity to the nucleotide sequence set forth in any one of SEQ ID NOs: 9-12;

[0100] As used herein, a Direct Repeat sequence can refer to the DNA encoding sequence in the CRISPR locus, or to the RNA encoded therefrom in a crRNA. Thus, when referring to any one of SEQ ID NOs: 9-12 in the context of an RNA molecule, such as a crRNA, each T is understood to represent a U.

[0101] In one embodiment, the Direct Repeat (DR) sequence comprises a nucleotide sequence having at least 80% identity to the nucleotide sequence set forth in any one of SEQ ID NOs: 9-12;

[0102] In one embodiment, the spacer sequence is more than 80% complementary to the target nucleic acid;

[0103] In one embodiment, the spacer sequence is more than 90%, more preferably more than 95%, even more preferably more than 99%, and even more preferably 100% complementary to the target nucleic acid;

[0104] In one embodiment, the spacer sequence is about or at least about 16 nucleotides in length, for example, about or at least about 16-100, about 16-90, about 17-70, about 17-50, about 18-41 contiguous nucleotides in length; optionally, wherein the spacer sequence is about 20 contiguous nucleotides in length;

[0105] In one embodiment, the spacer sequence is about 20 nt in length.

[0106] Target nucleic acid

[0107] As used herein, the term "target nucleic acid" is used interchangeably with "target sequence" or "target nucleic acid sequence" and refers to a particular nucleic acid that comprises a nucleic acid sequence that is fully or partially complementary to a spacer sequence in a guide RNA. A "target sequence" refers to a polynucleotide targeted by a spacer sequence in a guide RNA, e.g., a sequence that has complementarity to the spacer sequence, wherein hybridization between the target sequence and the spacer sequence will facilitate formation of a CRISPR-Cas complex (including a Cas protein and a guide RNA). Full complementarity is not required, so long as there is sufficient complementarity to cause hybridization and facilitate formation of a CRISPR-Cas complex. In some embodiments, the target nucleic acid comprises a non-coding region (e.g., a promoter or terminator). In some embodiments, the target nucleic acid is single-stranded, or double-stranded.

[0108] The target sequence can comprise any polynucleotide, such as DNA or RNA. In some cases, the target sequence is located within a cell or outside of a cell. In some cases, the target sequence is located within the nucleus, cytoplasm, organelle (e.g., mitochondrion or chloroplast) of a cell.

[0109] The target nucleic acid can be a sequence that encodes a gene product (e.g., a protein) or a non-coding sequence (e.g., a regulatory polynucleotide or junk DNA). In some cases, the target sequence should be associated with a protospacer adjacent motif (PAM).

[0110] In some embodiments, the target sequence comprises about or at least about 16 contiguous nucleotides of the target DNA, e.g., about or at least about 16-100, about 16-90, about 17-70, about 17-50 contiguous nucleotides of the target DNA; in some embodiments, wherein the target sequence comprises about 20 contiguous nucleotides of the target DNA.

[0111] In some embodiments, the reverse complement of the target sequence is immediately adjacent to the 3' end of a protospacer adjacent motif (PAM);

[0112] In some embodiments, the PAM is 5'-TTN, wherein N is A, T, G, or C.

[0113] cleavage

[0114] As used herein, "cleavage" refers to a DNA break in a target nucleic acid produced by a Cas protein described herein. In some embodiments, cleavage is a double-stranded DNA break. In some embodiments, cleavage is a single-stranded DNA break.

[0115] In the present disclosure, the meaning of cleaving a target nucleic acid or modifying a target nucleic acid can overlap. Modifying a target nucleic acid includes not only modification of a single nucleotide, but also insertion or deletion of a nucleic acid fragment.

[0116] reporter nucleic acid

[0117] As used herein, "reporter nucleic acid" refers to a molecule that can be cleaved or otherwise inactivated by an activated CRISPR system protein as described herein. The reporter nucleic acid comprises a nucleic acid element that can be cleaved by a CRISPR protein (e.g., employing a single-stranded non-targeting nucleic acid molecule comprising different reporter groups or label molecules at both ends). Cleavage of the nucleic acid element produces a detectable signal. Prior to cleavage, or when the reporter nucleic acid is in an "active" state, the reporter nucleic acid prevents the generation or detection of a positive detectable signal. It will be appreciated that in certain example embodiments, minimal background signal can be generated in the presence of an active reporter nucleic acid. The positive detectable signal can be any signal that can be detected using optical, fluorescent, chemiluminescent, electrochemical, or other detection methods known in the art. For example, in certain embodiments, a first signal (i.e., a negative detectable signal) can be detected when the reporter nucleic acid is present, which is then converted to a second signal (e.g., a positive detectable signal) upon detection of a target molecule and cleavage or inactivation by an activated CRISPR protein. The reporter nucleic acid can be a single-stranded DNA molecule, a single-stranded RNA molecule, or a single-stranded DNA-RNA hybrid.

[0118] The detection methods described herein can be used for quantitative detection of the target nucleic acid to be detected. The quantitative detection index described can be quantified according to the signal strength of the reporter group, such as the luminescence intensity of the fluorescent group, or the width of the color developing band, etc.

[0119] In one aspect, the disclosure also provides a method of detecting a target nucleic acid in a sample, the method comprising contacting the sample with a Cas protein as previously described, a guide RNA, and a non-target sequence; detecting a detectable signal resulting from cleavage of the non-target sequence by the Cas protein, thereby detecting the target nucleic acid; the non-target sequence does not hybridize to the guide RNA.

[0120] functional domain

[0121] As used herein, "functional domain" is taken in its broadest context to include a protein, e.g., an enzyme or factor, per se or a fragment / domain thereof having a particular function.

[0122] In some embodiments, the Cas protein (e.g., dCas protein) of the disclosure is linked / associated with one or more functional domains selected from one or more of a localization signal, a reporter protein, a Cas protein targeting moiety, a DNA binding domain, an epitope tag, a transcriptional activation domain, a transcriptional repression domain, a nuclease, a deaminase domain, a methylase, a demethylase, a transcriptional release factor, an HDAC, a lytic-activity polypeptide, a ligase. When more than one functional domain is included, the functional domains can be the same or different.

[0123] In one embodiment, the functional domain is selected from an adenosine deaminase catalytic domain or a cytidine deaminase catalytic domain.

[0124] In one embodiment, the localization signal comprises a nuclear localization signal and / or a nuclear export signal;

[0125] In some embodiments, the nuclear export signal comprises human protein tyrosine kinase 2;

[0126] In some embodiments, the reporter protein comprises one or more of glutathione-S-transferase, horseradish peroxidase, chloramphenicol acetyltransferase, beta-galactosidase, beta-glucuronidase, or a self-fluorescent protein;

[0127] In some embodiments, the self-fluorescent protein comprises one or more of green fluorescent protein, HcRed, DsRed, cyan fluorescent protein, yellow fluorescent protein, or blue fluorescent protein;

[0128] In some embodiments, the DNA binding domain comprises one or more of a methylation binding protein, LexA DBD, or Gal4 DBD;

[0129] In some embodiments, the epitope tag comprises one or more of a histidine tag, a V5 tag, a FLAG tag, an influenza virus hemagglutinin tag, a Myc tag, a VSV-G tag, or a thioredoxin tag;

[0130] In some embodiments, the transcription activation domain comprises VP64 and / or VPR;

[0131] In some embodiments, the transcription repression domain comprises KRAB and / or SID;

[0132] In some embodiments, the nuclease comprises Fokl;

[0133] In some embodiments, the deamination domain comprises one or more of ADARl, ADAR2, APOBEC, AID, or TAD;

[0134] In some embodiments, the cleavage-active polypeptide comprises a polypeptide having single-stranded RNA cleavage activity, a polypeptide having double-stranded RNA cleavage activity, a polypeptide having single-stranded DNA cleavage activity, or a polypeptide having double-stranded DNA cleavage activity;

[0135] In some embodiments, the ligase comprises a DNA ligase and / or an RNA ligase.

[0136] In some embodiments, the functional domain is a full-length or functional fragment of TadA8e.

[0137] Deaminase domain

[0138] As used herein, a “deaminase domain” includes a catalytic domain of a deaminase enzyme (e.g., an adenosine deaminase or a cytidine deaminase). As used herein, an “adenosine deaminase” or “adenosine deaminase protein” refers to a protein, polypeptide, or one or more functional domains of a protein or polypeptide that is capable of catalyzing a hydrolytic deamination reaction that converts an adenine (or an adenine moiety of a molecule) to a hypoxanthine (or a hypoxanthine moiety of a molecule), as shown below. In some embodiments, the adenine-containing molecule is adenosine (A), and the hypoxanthine-containing molecule is inosine (I). The adenine-containing molecule can be a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA).

[0139] Adenosine deaminases include, but are not limited to, members of the enzyme family known as adenosine deaminases that act on RNA (ADAR), members of the enzyme family known as adenosine deaminases that act on tRNA (ADAT), and other members of the family of adenosine deaminase domain (ADAD)-containing enzymes. According to the present disclosure, the adenosine deaminases are capable of targeting adenines in RNA / DNA and RNA duplexes. In particular embodiments, the adenosine deaminases have been modified to increase their ability to edit DNA in RNA / DNA heteroduplexes of RNA duplexes.

[0140] In some embodiments, the deaminase is a cytidine deaminase. The term “cytidine deaminase” or “cytidine deaminase protein” refers to a protein, polypeptide, or one or more functional domains of a protein or polypeptide that is capable of catalyzing a hydrolytic deamination reaction that converts a cytosine (or a cytosine moiety of a molecule) to a uracil (or a uracil moiety of a molecule). In some embodiments, the cytosine-containing molecule is cytidine (C), and the uracil-containing molecule is uridine (U). The cytosine-containing molecule can be a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA).

[0141] Cytidine deaminases include, but are not limited to, members of the enzyme family known as apolipoprotein B mRNA-editing complex (APOBEC) family deaminases, activation-induced deaminase (AID), or cytidine deaminase 1 (CDA1). In some embodiments, the cytidine deaminase includes an APOBEC family deaminase.

[0142] Identity

[0143] As used herein, "identity" is used to refer to the match of sequences between two polypeptides or between two nucleic acids, "identity" means the number of identical residues between the polypeptide or nucleic acid sequences divided by the total number of residues, and the calculation of the total number of residues is determined based on the type of mutation. The type of mutation includes insertion (extension) at either or both ends of the sequence, deletion (truncation) at either or both ends of the sequence, substitution / replacement of one or more amino acids / nucleotides, insertion within the sequence, and deletion within the sequence.

[0144] Taking the polypeptide sequence as an example, if the type of mutation is one or more of the following: substitution / replacement of one or more amino acids / nucleotides, insertion within the sequence, and deletion within the sequence, the total number of residues is calculated based on the larger one of the molecules being compared. If the type of mutation also includes insertion (extension) at either or both ends of the sequence or deletion (truncation) at either or both ends of the sequence, the number of amino acids inserted or deleted at either or both ends (e.g., less than 20 inserted or deleted at both ends) is not counted in the total number of residues. In calculating the percentage of identity, the sequences being compared are aligned in a manner that produces the maximum match between the sequences, and gaps in the alignment, if any, are addressed by a particular algorithm. The calculation of identity of nucleotides is the same.

[0145] Vector

[0146] As used herein, a "vector" is a nucleic acid molecule capable of transporting another nucleic acid molecule to which it has been linked.

[0147] Vectors include, but are not limited to, single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules that include one or more free ends, no free ends (e.g., circular); nucleic acid molecules that include DNA, RNA, or both; and other diverse polynucleotides known in the art. A vector can be introduced into a host cell by transformation, transduction, or transfection, such that the genetic material elements it carries are expressed in the host cell. A vector can be introduced into a host cell to thereby produce a transcript, protein, or peptide, including from a protein, fusion protein, isolated nucleic acid molecule, etc. (e.g., a CRISPR transcript, such as a nucleic acid transcript, protein, or enzyme) as described herein. A vector can contain a variety of control elements that regulate expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. A vector can also contain a replication origin.

[0148] Vectors include plasmids, which refer to circular double stranded DNA loops that are used as carriers for replication of inserted foreign DNA in host cells, and viral vectors, which refer to viral genomes or portions thereof used as vectors for replication of inserted foreign DNA in host cells. Viral vectors include, for example, retroviruses, replication-defective retroviruses, adenoviruses, replication-defective adenoviruses, and adeno-associated viruses. Viral vectors also include polynucleotides carried by a virus for transfection into a host cell. Some vectors (for example, bacterial vectors with a bacterial origin of replication and episomal mammalian vectors) are capable of autonomous replication in a host cell into which they are introduced.

[0149] Other vectors (for example, non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing expression of genes to which they are operatively-linked. Such vectors are referred to as "expression vectors."

[0150] Those of skill in the art will appreciate that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of

[0151] Regulatory elements

[0152] As used herein, "regulatory elements" include promoters, enhancers, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals such as polyadenylation signals, poly-U sequences), which are described in detail in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif (1990). In some cases, regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host cells as well as those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). Tissue-specific promoters can direct expression primarily in a desired tissue of interest, such as muscle, neuronal, bone, skin, blood, a particular organ (e.g., liver, pancreas), or a particular cell type (e.g., lymphocytes). In other cases, regulatory elements can also direct expression in a temporal-dependent manner (e.g., in a cell cycle-dependent or developmental stage-dependent manner), which can or can not be tissue- or cell type-specific.

[0153] A "promoter" refers to a nucleotide sequence located upstream of a gene that initiates transcription of the downstream gene. A constitutive promoter is a nucleotide sequence that, when operably linked with a polynucleotide encoding or specifying a gene product, will result in the production of the gene product in a cell under most or all physiological conditions of the cell. An inducible promoter refers to a promoter that selectively expresses a coding sequence or functional RNA in response to the presence of an endogenous or exogenous stimulus, e.g., by a chemical compound (chemical inducer), or in response to an environmental, hormonal, chemical, and / or developmental signal. Inducible or regulated promoters include, for example, promoters that are induced or regulated by light, heat, stress, flooding or drought, salt stress, osmotic stress, plant hormones, wounding, or chemicals such as ethanol, abscisic acid (ABA), jasmonates, salicylic acid, or safeners.

[0154] Host cell

[0155] As used herein, a "host cell" refers to a eukaryotic cell (e.g., an animal cell, a plant cell, a fungal cell, etc.), a prokaryotic cell (e.g., some microbial cell, E. coli, Bacillus, etc.), or a cell from a multicellular organism (e.g., a cell line) cultured in a unicellular entity, which serves as a recipient for a nucleic acid (e.g., an expression vector), and includes the progeny of the original cell that has been genetically modified by the nucleic acid.

[0156] It is understood that progeny of a single cell can not necessarily be completely identical to the original parent cell in morphology or genome, etc., due to natural, accidental, or deliberate mutation. A "recombinant host cell" (also referred to as a "genetically modified host cell") is a host cell in which a heterologous nucleic acid, e.g., an expression vector, has been introduced.

[0157] In one aspect, the disclosure provides a host cell or progeny thereof, the host cell comprising a Cas protein of the disclosure, or a fusion protein of the disclosure, or a polynucleotide of the disclosure, or a CRISPR-Cas system / composition of the disclosure.

[0158] In some embodiments, the cell or progeny thereof comprises a modification that is not present in its wild type.

[0159] In some embodiments, the host cell is from a plant or an animal.

[0160] In some embodiments, the plant is a dicot. In some embodiments, the dicot is selected from the group consisting of soybean, cabbage (e.g., Chinese cabbage), rapeseed, Brassica, watermelon, melon, potato, tomato, tobacco, eggplant, pepper, cucumber, cotton, alfalfa, eggplant, grape.

[0161] In some embodiments, the plant is a monocot, illustratively, the monocot is selected from the group consisting of rice, maize, wheat, barley, oat, sorghum, millet, grasses, Poaceae, Zizania, Avena, Coix, Hordeum, Oryza, Panicum (e.g., Panicum miliaceum), Secale, Setaria (e.g., Setaria italica), Sorghum, Triticum, Zea, Cymbopogon, Saccharum (e.g., Saccharum officinarum), Phyllostachys, Dendrocalamus, Bambusa, Yushania.

[0162] In some embodiments, the animal is selected from a pig, an ox, a sheep, a goat, a mouse, a rat, a llama, a monkey, a rabbit, a chicken, a duck, a goose, a fish (e.g., a zebrafish). In some embodiments, the cell is a eukaryotic cell, injected into a mammalian cell, including a human cell (primary human cells or an established human cell line). In some embodiments, the cell is a non-human mammalian cell, such as a cell from a non-human primate (e.g., a monkey), a cow / bull / cattle, a sheep, a goat, a pig, a horse, a dog, a cat, a rodent (such as a rabbit, a mouse, a rat, a hamster, etc.). In some embodiments, the cell is from a fish (such as a salmon), a bird (such as an avian, including a chicken, a duck, a goose), a reptile, a shellfish (e.g., an oyster, a clam, a lobster, a prawn), an insect, a worm, a yeast, etc. In some embodiments, the cell is from a plant, e.g., a monocot or a dicot. In certain embodiments, the plant is a food crop, such as barley, cassava, cotton, groundnut or peanut, maize, millet, oil palm fruit, potato, dry bean, rapeseed or canola, rice, rye, sorghum, soybean, sugarcane, sugar beet, sunflower, and wheat. In certain embodiments, the plant is a cereal (barley, maize, millet, rice, rye, sorghum, and wheat). In certain embodiments, the plant is a tuber (cassava and potato). In certain embodiments, the plant is a sugar crop (sugar beet and sugarcane). In certain embodiments, the plant is an oil crop (soybean, groundnut or peanut, rapeseed or canola, sunflower, and oil palm fruit). In certain embodiments, the plant is a fiber crop (cotton). In certain embodiments, the plant is a tree (such as a peach or nectarine tree, an apple or pear tree, a nut tree (such as an almond or walnut or pistachio tree), or a citrus tree (e.g., an orange, a grapefruit, or a lemon tree)), a grass, a vegetable, a fruit, or an alga. In certain embodiments, the plant is a Solanum plant; a Brassica plant; a Lactuca plant; a Spinacia plant; a Capsicum plant; cotton, tobacco, asparagus, carrot, cabbage, broccoli, cauliflower, tomato, eggplant, pepper, lettuce, spinach, strawberry, blueberry, raspberry, blackberry, grape, coffee, cocoa, etc.

[0163] In one aspect, the disclosure provides a multicellular organism comprising a host cell of the disclosure or progeny thereof.

[0164] In some embodiments, the multicellular organism is an animal model or a plant model for a related disease.

[0165] NLS

[0166] As used herein, “NLS” refers to “nuclear localization sequence” or “nuclear localization signal” and refers to an amino acid sequence that facilitates entry of a protein into the nucleus of a cell. Nuclear localization sequences are known in the art (e.g., described in International PCT Application PCT / EP2000 / 011690 filed November 23, 2000 by Plank et al. and published as WO / 2001 / 038547 on May 31, 2001), which is incorporated herein by reference for its disclosure of exemplary nuclear localization sequences. In other embodiments, the NLS is an optimized NLS, e.g., as described in Koblan et al., Nature Biotech. 2018 doi:10.1038 / nbt.4172. In some embodiments, the NLS comprises the following amino acid sequence: KRTADGSEFESPKKKRKV (SEQ ID NO:30), AVKRPAATKKAGQAKKKKLD (SEQ ID NO:31), KRPAATKKAGQAKKKK (SEQ ID NO:32), KKTELQTTNAENKTKKL (SEQ ID NO:33), KRGINDRNFWRGENGRKTR (SEQ ID NO:34), RKSGKIAAIVVKRPRK (SEQ ID NO:35), PKKKRKV (SEQ ID NO:36), or MDSLLMNRRKFLYQFKNVRWAKGRRETYLC (SEQ ID NO:37).

[0167] operably linked

[0168] As used herein, “operably linked” refers to a target nucleotide sequence linked to a regulatory element in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell). Advantageous vectors include lentivirus and adeno-associated virus, and the type of these vectors can also be selected to target specific types of cells.

[0169] complementary

[0170] As used herein, "complementarity" refers to the capacity of one nucleic acid sequence to form one or more hydrogen bonds with another nucleic acid sequence by virtue of traditional Watson-Crick or other non-traditional types of bonding. Percent complementarity indicates the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 complementary, then the percent complementarity is 50%, 60%, 70%, 80%, 90%, and 100%). "Perfect complementarity" indicates that all consecutive residues of one nucleic acid sequence form hydrogen bonds with the same number of consecutive residues in another nucleic acid sequence. "Substantial complementarity" refers to a degree of complementarity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% in a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refers to hybridization under stringent conditions.

[0171] As used herein, the term "stringent conditions" in relation to hybridization refers to conditions under which a nucleic acid having complementarity to a target sequence will hybridize primarily to that target sequence and not to non-target sequences. Stringent conditions are often sequence dependent, and are dependent on many factors. In general, the longer the sequence, the higher the temperature at which the sequence will specifically hybridize to its target sequence.

[0172] As used herein, "hybridization" refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via the hydrogen bonding between the bases of the nucleotides residues. The complex can comprise two strands forming a duplex, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination of these. A hybridization reaction can constitute a step in a more extensive process such as the initiation of PCR, or cleavage of a polynucleotide by an enzyme. A sequence capable of hybridizing to a given sequence is referred to as the "complement" of the given sequence.

[0173] Hybridization of a target sequence to a gRNA means that at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the nucleic acid sequences of the target sequence and the gRNA can hybridize to form a complex; or means that at least 12, 15, 16, 17, 18, 19, 20, or more bases of the nucleic acid sequences of the target sequence and the gRNA can base pair to hybridize to form a complex.

[0174] Methods of modification

[0175] The CRISPR-Cas compositions / systems of the present disclosure comprising the Cas proteins of the present disclosure have a variety of uses, including editing / modifying (e.g., cleaving, deleting, inserting, translocating, inactivating, or activating) a target DNA in a variety of cell types. The CRISPR-Cas systems / compositions herein have a wide range of applications, such as drug screening, disease diagnosis and prognosis, and treatment of various genetic diseases.

[0176] In one aspect, the present disclosure provides a method of targeting and editing a target nucleic acid, the method comprising contacting the target nucleic acid with a CRISPR-Cas system or composition of the present disclosure.

[0177] In yet another aspect, the present disclosure also provides a method of non-specifically degrading single-stranded DNA upon recognizing a target nucleic acid, the method comprising contacting the target nucleic acid with the aforementioned CRISPR-Cas composition.

[0178] In yet another aspect, the present disclosure also provides a method of targeting and cleaving a single-stranded target nucleic acid, the method comprising contacting the target nucleic acid with a CRISPR-Cas system / composition of the present disclosure.

[0179] In yet another aspect, the present disclosure also provides a method of inducing a change in a cell state, the method comprising contacting a CRISPR-Cas composition of the present disclosure with the target nucleic acid in the cell.

[0180] In one embodiment, the cell state comprises apoptosis or dormancy;

[0181] In one embodiment, the cell comprises a eukaryotic cell or a prokaryotic cell;

[0182] In one embodiment, the cell comprises a mammalian cell or a plant pathogenic cell;

[0183] In one embodiment, the cell comprises a cancer cell;

[0184] In one embodiment, the cell comprises an infectious cell or a cell infected by an infectious agent;

[0185] In one embodiment, the cell comprises a cell infected by a virus, a cell infected by a prion;

[0186] In one embodiment, the cell comprises a cell infected by a virus, a cell infected by a prion;

[0187] In one embodiment, the cell comprises a fungal cell, a protozoan or a parasitic cell.

[0188] The methods and / or systems of the present disclosure can be used to modify a target DNA, for example, to modify translation and / or transcription of one or more genes of a cell. For example, the modification can result in an increase in transcription / translation / expression of a gene. In other embodiments, the modification can result in a decrease in transcription / translation / expression of a gene.

[0189] In yet another aspect, the present disclosure also provides a method of obtaining a plant with a desired trait, using the aforementioned gene editing system to contact a plant cell, to modify a gene of the plant cell or to introduce a desired gene into the plant cell, the modification or the desired gene being capable of expressing the desired trait, to obtain a modified plant cell,

[0190] using the modified plant cell to regenerate, to obtain the plant with the desired trait.

[0191] In one aspect, the present disclosure also provides a method of identifying a desired trait in a plant, a desired gene in a plant cell being capable of expressing the desired trait, using the aforementioned CRISPR-Cas composition to contact the plant cell, to identify the desired gene.

[0192] In one aspect, the present disclosure also provides an implantable device, the implantable device comprising the aforementioned CRISPR-Cas composition.

[0193] In one embodiment, the CRISPR-Cas system or composition is within a matrix.

[0194] In one embodiment, the CRISPR-Cas system or composition is within a depot.

[0195] Pharmaceutical composition

[0196] In yet another aspect, the present disclosure provides a pharmaceutical composition comprising the system / composition of the present disclosure, the vector of the present disclosure or the cell of the present disclosure; and a pharmaceutically acceptable excipient.

[0197] In some embodiments, the pharmaceutical composition is an injection.

[0198] Treatment

[0199] As used herein, "treatment" refers to treating or curing a subject's condition, delaying the onset of symptoms of a condition, and / or delaying the severity of a condition. The term "subject" includes, but is not limited to, various animals, plants, and microorganisms. Animals, including mammals, such as bovids, equids, ovines, suids, canids, felids, leporids, rodents (e.g., mice or rats), non-human primates (e.g., macaques or cynomolgus monkeys), or humans. In certain embodiments, the subject (e.g., human) has a condition (e.g., a condition resulting from a disease-associated gene defect). A "plant" is any differentiated multicellular organism capable of photosynthesis, including crop plants at any stage of maturation or development.

[0200] In yet another aspect, the disclosure provides a method for diagnosing, preventing, or treating a disease in a subject in need thereof, the method comprising administering to the subject a Cas protein of the disclosure, a fusion protein of the disclosure, a system / composition of the disclosure, a host cell of the disclosure, or a pharmaceutical composition of the disclosure, wherein the disease is associated with a target DNA, wherein the spacer sequence is capable of hybridizing to a target sequence of the target DNA, wherein the target DNA is modified by the complex, and wherein the modification of the target DNA diagnoses, prevents, or treats the disease.

[0201] In one embodiment, the use comprises administering the CRISPR-Cas composition to the subject or to an ex vivo cell of the subject;

[0202] In one embodiment, the spacer sequence is complementary to at least 15 nucleotides of the target nucleic acid associated with the condition or disease, the Cas protein targets and / or cleaves the target nucleic acid;

[0203] In one embodiment, the condition or disease comprises a cancer or an infectious disease;

[0204] In one embodiment, the cancer comprises one or more of Wilms' tumor, Ewing's sarcoma, neuroendocrine tumor, glioblastoma, neuroblastoma, melanoma, skin cancer, breast cancer, colon cancer, rectal cancer, prostate cancer, liver cancer, kidney cancer, pancreatic cancer, lung cancer, biliary tract cancer, cervical cancer, endometrial cancer, esophageal cancer, stomach cancer, head and neck cancer, medullary thyroid cancer, ovarian cancer, glioma, lymphoma, leukemia, myeloma, acute lymphoblastic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, or urinary bladder cancer;

[0205] In an embodiment, the disorder or disease comprises one or more of cystic fibrosis, Duchenne Muscular Dystrophy, Becker Muscular Dystrophy, alpha-1-antitrypsin deficiency, Pompe disease, myotonic muscular dystrophy, Huntington’s disease, fragile X syndrome, Friedreich’s ataxia, amyotrophic lateral sclerosis, frontotemporal dementia, hereditary chronic kidney disease, hyperlipidemia, hypercholesterolemia, Leber congenital amaurosis, sickle cell disease, hypercholesterolemia, atherosclerosis, primary hyperoxaluria (PH1), hereditary angioedema (HAE), transthyretin amyloidosis, or beta thalassemia.

[0206] In an embodiment, the infectious agent of the infectious disease comprises one or more of human immunodeficiency virus, herpes simplex virus-1, Hepatitis B Virus, or herpes simplex virus-2.

[0207] Kit

[0208] In yet another aspect, the disclosure provides a kit comprising a Cas protein of the disclosure, a polynucleotide of the disclosure, a system / composition of the disclosure, a host cell of the disclosure, or a pharmaceutical composition of the disclosure, or any one, two, or all components thereof.

[0209] In some embodiments, the kit of the disclosure further comprises instructions for using one or more components contained therein, and / or instructions for combining with additional component(s) that can be obtained or necessary elsewhere.

[0210] In some embodiments, the kit further comprises one or more buffers that can be used to solubilize any of the one or more components contained therein, and / or to provide suitable reaction conditions for one or more of the one or more components. Such buffers can include one or more of PBS, HEPES, Tris, MOPS, Na2CO3, NaHCO3, NaB, or combinations thereof. In some embodiments, the reaction conditions include an appropriate pH, such as an alkaline pH. In some embodiments, the pH is between 7-10.

[0211] In some embodiments, any one or more of the kit components can be stored in a suitable container or at a suitable temperature, e.g., 4 degrees Celsius.

[0212] In yet another aspect, the disclosure provides a container comprising the kit of the disclosure.

[0213] In an embodiment, the container comprises a sterile container; in an embodiment, the container comprises a syringe.

[0214] Expression

[0215] Nucleic acid expression includes one or more of production of RNA templates from DNA sequences (e.g., transcription), processing of RNA transcripts (e.g., by splicing, editing, 5' cap formation, and / or 3' end processing), translation of RNA into polypeptides or proteins, or post-translational modification of polypeptides or proteins.

[0216] Delivery

[0217] As used herein, “delivery” refers to providing an entity (e.g., a drug) to a destination, e.g., components of the CRISPR-Cas system / compositions of the present disclosure can be delivered in various forms, e.g., DNA / RNA or RNA / RNA or protein RNA combinations. For example, Cas proteins can be delivered as polynucleotides encoding DNA or polynucleotides encoding RNA or as proteins.

[0218] In one aspect, the present disclosure provides a delivery system comprising the Cas protein or the fusion protein, or the polynucleotide, or the CRISPR-Cas composition and a delivery vehicle.

[0219] In one embodiment, the delivery system further comprises a delivery vehicle, the delivery vehicle comprising a nanoparticle, a plasmid, an exosome, a microvesicle, a gene gun, or an electroporation device.

[0220] Linker

[0221] As used herein, “linker” refers to a linear polypeptide formed by the linkage of multiple amino acid residues through peptide bonds. The linker can be selected from an artificially synthesized amino acid sequence or a naturally occurring polypeptide sequence.

[0222] Examples

[0223] Materials and Methods

[0224] Experimental methods used in the examples are routine unless otherwise specified. Materials, reagents, etc. used in the examples are commercially available unless otherwise specified. The following materials and experimental methods were used in the examples unless otherwise specified.

[0225] Example 1 Obtaining Cas proteins

[0226] The inventors performed analysis on metagenome of uncultured microorganism, and identified 4 new Cas proteins through de-redundancy, protein clustering, etc. Blast analysis results showed that the Cas proteins had low sequence similarity with the reported Cas proteins, and the Cas proteins were named YTGE-167, YTGE-174, YTGE-190 and YTGE-192 in the present disclosure.

[0227] The amino acid sequences of the above-mentioned Cas proteins are shown in SEQ ID NOs: 1-4, respectively, and the nucleic acid sequences encoding the same are shown in SEQ ID NOs: 5-8. The direct repeat (DR) sequences of the guide RNA corresponding to the above-mentioned proteins were analyzed, and the DR sequences of each Cas protein were determined as shown in SEQ ID NOs: 9-12 (in the description of the DR sequence, any "T" in the sequence refers to "U"). The inventors further analyzed the RNA secondary structure of each DR sequence using RNAfold. The analysis results are shown in FIG. 2. Through any embodiment of the PAM library depletion experiment (the experiment is performed according to Karvelis et al. Methods. 2017 May 15; 121-122: 3-8 (the entire contents of which are incorporated herein by reference)), it was found that the PAM corresponding to each of the above-mentioned Cas proteins was 5'-TTN.

[0228] It is identified that the Cas protein of the present disclosure belongs to the Cas12 family.

[0229] Example 2 Verification of Cas protein cleavage activity

[0230] 1. Plasmid construction

[0231] (1) Taking the TTR gene target as the targeting object, the spacer sequence TTR-spacer (SEQ ID NO: 13) was designed, and the TTR-crRNA sequence of each Cas protein was designed as shown in the following table (the structure of the crRNA sequence is 5'-DR sequence-spacer sequence-3').

[0232] Table 1

[0233] The coding nucleotides (SEQ ID NOs: 5-8) of YTGE-167, YTGE-174, YTGE-190 and YTGE-192 and the corresponding TTR-crRNA were cloned into pcDNA3.1-HA (Addgene, #128034), and the crRNA was regulated by T7 promoter to obtain a recombinant expression plasmid: T7-Cas-T7-crRNA vector (the vector map is shown in FIG. 1).

[0234] (2) The araC-pBAD-CCDB fragment (SEQ ID NO: 18) with TTR target sequence (the sequence is the same as the spacer TTR-spacer, see SEQ ID NO: 13) was synthesized by Suzhou Hongxun Biotechnology Co., Ltd., and the araC-pBAD-CCDB fragment was inserted into the 1284-1300 site of the pKESK22 (Addgene, Plasmid #64857) plasmid to obtain the Target plasmid. The sequence of the Target plasmid is shown in SEQ ID NO: 19, and the plasmid map is shown in FIG. 3.

[0235] 2. Preparation and transformation of E. coli competent cells

[0236] The Target plasmid was transformed into DH5a competent cells, and the streaked inoculation was inoculated on LB solid medium containing 50 μg / ml kanamycin sulfate. It was placed in a biochemical incubator at 37°C overnight, and the next day a single colony was picked from the plate and inoculated into an LB liquid medium test tube containing 4 ml of 50 μg / ml kanamycin sulfate (Shenguo Biotechnology, A100408-0100). The culture was incubated at 37°C, 200 rpm overnight. The next day, 4 ml of bacterial solution was inoculated into a 2L flask containing 400 ml of LB liquid medium with 50 μg / ml kanamycin sulfate, and the culture was incubated at 37°C, 200 rpm for 2-3 hours.

[0237] When the OD600nm value of the bacterial solution reached 0.3-0.5, the flask was removed and placed on ice for 10-15 min. Under sterile conditions, the bacterial solution was poured into a pre-cooled 500 ml centrifuge bottle, centrifuged at 4°C, 3000 rpm for 8 min, the supernatant was discarded, about 200 ml of pre-cooled CaCl2 solution was added, and the bacterial suspension was mixed by blowing and placed in an ice bath for 30 min. Then the bacterial solution was centrifuged at 4°C, 3000 rpm for 8 min, the supernatant was discarded, about 8 ml of pre-cooled CaCl2 solution was added, the bacterial suspension was resuspended, and the resuspended bacterial suspension was aliquoted into 1.5 ml EP tubes, 110 μl per tube, and stored in a -80°C ultra-low temperature refrigerator for use.

[0238] 3. In vivo editing efficiency determination of E. coli

[0239] 100ng T7-Cas-T7-crRNA vector was transformed into E. coli competent cells, and after recovering for half an hour, it was plated on plates added with carbenicillin disodium (Shenguo Biotech, A100358-0001) and L-arabinose (Shenguo Biotech, A610071-0100) (CL) and kanamycin sulfate (Shenguo Biotech, A100408-0100) and carbenicillin disodium (Shenguo Biotech, A100358-0001) (CK), and cultured overnight for 16 h. If the Cas protein cleaves the target plasmid, the bacteria survive. The number of colonies on the CL plate / the number of colonies on the CK plate is the editing efficiency. The editing efficiency of each Cas protein was detected in the above manner, and the results are shown in FIG. 4 (where the plate count under the numbers "167", "174", "192" and "190" represents the cleavage of YTGE-167, YTGE-174, YTGE-190 and YTGE-192, respectively). Analysis shows that each Cas protein, such as YTGE-167, YTGE-174, YTGE-190 and YTGE-192, has significant cleavage activity and has potential application prospects.

[0240] Targeting of each nuclease to mammalian cells

[0241] To evaluate the cleavage activity of each nuclease in mammalian cells, the applicant cloned each nuclease (SEQ ID NO: 5-8), such as YTGE-167, YTGE-174, YTGE-190 and YTGE-192, into the pcDNA3.1 (Invitrogen, V79020) backbone to obtain a nuclease expression plasmid. The HAO1 gene and the TTR gene were selected as targets, and the target sequences are shown in the following table:

[0242] Table 2

[0243] Each corresponding crRNA sequence was designed, as shown in the following table:

[0244] Table 3

[0245] The crRNA was cloned into the pUC19 vector (NEB, N3041L) and regulated by the U6 promoter to obtain a crRNA expression plasmid. Then, each constructed plasmid was transfected into E. coli DH5a competent cells (Weidi Biotech, DL1001), amplified in large quantities, and then the concentration was determined and stored in a -20°C refrigerator for standby use.

[0246] HEK293T cells (purchased from ATCC) were seeded in DMEM medium (Gibco, 11965092) added with 10% FBS (v / v) containing 1% Penicillin Streptomycin (v / v) (Gibco, 15140122) and cultured in a 37°C cell incubator containing 5% CO2. Cells for transfection were seeded in 24-well cell culture plates the day before and observed the next day. When the cells grew to about 80% of the cell density, transfection was performed.

[0247] The nuclease expression plasmid, the corresponding crRNA expression plasmid and the EGFP-C1 (Addgene, Plasmid, #54759) plasmid were transfected into HEK293T cells respectively. The amount of plasmid transfected into cells in each well of the 24-well plate was 0.3 μg of nuclease expression plasmid, 0.3 μg of crRNA expression plasmid and 0.3 μg of EGFP-C1 plasmid. The specific transfection operation was as follows:

[0248] The nuclease expression plasmid, the crRNA expression plasmid and the EGFP-C1 plasmid were mixed and diluted with 25 μl of serum-reduced medium (Source Bioscience, L530KJ) for transfection, and then 2 μl of Lipofectamine 3000 (Invitrogen, L3000015) reagent was added and mixed to prepare reagent A, which was allowed to stand for 5 minutes. The nuclease expression plasmid, the crRNA expression plasmid and the EGFP-C1 plasmid were mixed and diluted with 25 μl of serum-reduced medium (Source Bioscience, L530KJ) for transfection, and then 2 μl of Lipofectamine 3000 (Invitrogen, L3000015) reagent was added and mixed to prepare reagent A, which was allowed to stand for 5 minutes. The nuclease expression plasmid, the crRNA expression plasmid and the EGFP-C1 plasmid were mixed and diluted with 25 μl of serum-reduced medium (Source Bioscience, L530KJ) for transfection, and then 2 μl of Lipofectamine 3000 (Invitrogen, L3000015) reagent was added and mixed to prepare reagent A, which was allowed to stand for 5 minutes.

[0249] The above reagent A and reagent B were mixed and uniformly blown, and allowed to stand for 20 minutes. After standing, the mixed reagent was added dropwise to the 24-well plate cells to be transfected, and then placed back into a 37°C, 5% CO2 incubator for culture. After 6 hours of transfection, the culture medium was replaced with DMEM medium containing 10% FBS.

[0250] After 48 hours of transfection, the expression of EGFP fluorescent protein indicated that the cells were successfully transfected, and the cells positive for EGFP expression were sorted for detection of editing efficiency. The cells were subjected to genomic extraction (using a genomic DNA extraction kit, TIANGEN, DP304-03). The PCR amplified product was used for high-throughput deep sequencing (Kangke Biological Technology Co., Ltd.), and the cutting activity was detected. The detection results are shown in FIG. 5.

[0251] From the analysis of the examples, it can be known that each of the nucleases of the present disclosure has significant cleavage activity in mammalian cells, and has potential application prospects. Since the nucleases of the present disclosure also have the characteristics of small size and short crRNA, they are suitable for in vivo multiplex gene editing applications, such as AAV or LNP, and have great potential for therapeutic gene editing applications, and can be used for in vivo or ex vivo therapeutic applications in the future.

[0252] Part of the sequence information involved herein is as follows:

[0253] Although the present disclosure has been described with reference to specific embodiments thereof, it will be understood by those skilled in the art that various changes can be made and equivalents can be substituted without departing from the true spirit and scope of the present disclosure. In addition, many modifications can be made to adapt specific situations, materials, compositions of matter, methods, one or more method steps to the purpose, spirit and scope of the present disclosure. All such modifications are intended to be within the scope of the appended claims.

Claims

1. A Cas protein, characterized in that, The protein is selected from the group consisting of: (a) a polypeptide having an amino acid sequence as set forth in any one of SEQ ID NOs: 1-4; (b) a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% homology (or identity) to an amino acid sequence as set forth in any one of SEQ ID NOs: 1-4, and the polypeptide has a biological function as set forth in any one of SEQ ID NOs: 1-4; (c) a derivative polypeptide formed by substitution, deletion, or addition of one or more (preferably 1-20, more preferably 1-10, most preferably 1-5) amino acid residues to an amino acid sequence as set forth in any one of SEQ ID NOs: 1-4, and the derivative polypeptide retains a biological function as set forth in any one of SEQ ID NOs: 1-4.

2. A fusion protein, characterized in that, The fusion protein comprises the Cas protein of claim 1 and one or more functional domains; Optionally, the functional domain is selected from one or more of a localization signal, a reporter protein, a Cas protein targeting moiety, a DNA binding domain, an epitope tag, a transcription activation domain, a transcription repression domain, a nuclease, a deamination domain, a methylase, a demethylase, a transcription release factor, an HDAC, a cleavage active polypeptide, a ligase; Optionally, the functional domain is selected from an adenosine deaminase catalytic domain or a cytidine deaminase catalytic domain; Optionally, the localization signal comprises a nuclear localization signal and / or a nuclear export signal; Optionally, the nuclear export signal comprises human protein tyrosine kinase 2; Optionally, the reporter protein comprises one or more of glutathione-S-transferase, horseradish peroxidase, chloramphenicol acetyltransferase, beta-galactosidase, beta-glucuronidase, or a self-fluorescent protein; Optionally, the self-fluorescent protein comprises one or more of green fluorescent protein, HcRed, DsRed, cyan fluorescent protein, yellow fluorescent protein, or blue fluorescent protein; Optionally, the DNA binding domain comprises one or more of a methylation binding protein, LexA DBD, or Gal4 DBD; Optionally, the epitope tag comprises one or more of a histidine tag, a V5 tag, a FLAG tag, an influenza virus hemagglutinin tag, a Myc tag, a VSV-G tag, or a thioredoxin tag; Optionally, the transcription activation domain comprises VP64 and / or VPR; Optionally, the transcription repression domain comprises KRAB and / or SID; Optionally, the nuclease comprises Fokl; Optionally, the deamination domain comprises one or more of ADAR1, ADAR2, APOBEC, AID, or TAD. Optionally, the cleavage- active polypeptide comprises a polypeptide having single- stranded RNA cleavage activity, a polypeptide having double- stranded RNA cleavage activity, a polypeptide having single- stranded DNA cleavage activity, or a polypeptide having double- stranded DNA cleavage activity; Optionally, the ligase comprises a DNA ligase and / or an RNA ligase; Optionally, the functional domain is a full length or a functional fragment of TadA8e.

3. A polynucleotide, comprising, The polynucleotide is a nucleotide sequence encoding the Cas protein of claim 1 or the fusion protein of claim 2, or a nucleotide sequence encoding the fusion protein of claim 2; Optionally, the polynucleotide is a DNA molecule codon-optimized according to the codon bias of the host cell; Optionally, the host cell comprises a prokaryotic cell or a eukaryotic cell; Optionally, the DNA molecule comprises a nucleotide having 70% or more (optionally, 90% or more, optionally, 95% or more, optionally, 99% or more, optionally, 100%) identity to the nucleotide sequence of any one of SEQ ID NOs: 5-8.

4. A CRISPR-Cas composition, characterized in that, The composition comprises: (1) the Cas protein of claim 1 or the fusion protein of claim 2, or a nucleic acid molecule encoding the Cas protein of claim 1 or the fusion protein of claim 2; (2) a guide RNA, or one or more DNA molecules encoding the guide RNA, the guide RNA comprising: (i) a direct repeat (DR) sequence capable of forming a complex with the Cas12i polypeptide; and (ii) a spacer sequence capable of hybridizing to a target sequence of a target DNA, thereby directing the complex to the target DNA; Optionally, wherein the direct repeat sequence is at the 5’ end of the spacer sequence; Optionally, the direct repeat sequence has a secondary structure substantially identical to the secondary structure of any one of SEQ ID NOs: 9-12; Optionally, wherein the direct repeat sequence: (1) comprises the polynucleotide sequence of any one of SEQ ID NOs: 9-12; or (2) comprises a polynucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity to any one of SEQ ID NOs: 9-12; Optionally, the target sequence comprises about or at least about 16 contiguous nucleotides of the target DNA, for example, about or at least about 16-100, about 16-90, about 17-70, about 17-50 contiguous nucleotides of the target DNA; optionally, wherein the target sequence comprises about 20 contiguous nucleotides of the target DNA; Optionally, the reverse complement of the target sequence is immediately adjacent to the 3’ end of a protospacer adjacent motif (PAM); optionally, the PAM is 5’-TTN, wherein N is A, T, G, or C; Optionally, the spacer sequence is about or at least about 16 nucleotides in length, e.g., about or at least about 16-100, about 16-90, about 17-70, about 17-50, about 18-41 contiguous nucleotides in length; optionally, wherein the spacer sequence is about 20 contiguous nucleotides in length. Optionally, the target DNA is a dsDNA, such as a eukaryotic dsDNA, e.g., a gene in a eukaryotic cell.

5. The CRISPR-Cas composition of claim 4, comprising one or more vectors comprising: (1) a first regulatory element operably linked to a nucleotide sequence encoding the Cas protein or a nucleotide sequence encoding the fusion protein; and (2) a second regulatory element operably linked to a nucleotide sequence encoding the guide RNA, the guide RNA comprising: (a) a spacer sequence capable of hybridizing to a target sequence of a target nucleic acid; and (b) a Direct Repeat (DR) sequence linked to the spacer sequence capable of directing the Cas protein to bind to the guide RNA to form a CRISPR-Cas complex targeting the target sequence; wherein the first regulatory element and the second regulatory element are on the same or different vectors of the CRISPR-Cas vector system.

6. A delivery system characterized by, the delivery system comprises the Cas protein of claim 1, or the fusion protein of claim 2, or the polynucleotide of claim 3, or the CRISPR-Cas composition of any one of claims 4-5, and a delivery vehicle; Optionally, the delivery vehicle comprises a nanoparticle, a plasmid, an exosome, a microvesicle, a gene gun, or an electroporation device.

7. A host cell or progeny thereof, wherein, the host cell comprises the Cas protein of claim 1, or the fusion protein of claim 2, or the polynucleotide of claim 3, or the CRISPR-Cas composition of any one of claims 4-5, or the delivery system of claim 6; Optionally, the cell or progeny thereof comprises a modification that is not present in its wild type; Optionally, the host cell is from a plant or an animal.

8. A method of targeting and editing a target nucleic acid, characterized in that, the method comprises contacting the target nucleic acid with the Cas protein of claim 1, or the fusion protein of claim 2, or the polynucleotide of claim 3, or the CRISPR-Cas composition of any one of claims 4-5, or the host cell of claim 7.

9. A method of non-specifically degrading or cleaving single-stranded nucleic acids upon recognition of a target nucleic acid, characterized in that, the method comprises contacting a target sequence of the target nucleic acid with the CRISPR-Cas composition of any one of claims 4-5.

10. A method of targeting and cleaving a double-stranded target nucleic acid, characterized in that, the method comprises contacting the double-stranded target nucleic acid with the CRISPR-Cas composition of any one of claims 4-5.

11. A method of inducing a change in the state of a cell, characterized by, the method comprises contacting the CRISPR-Cas composition of any one of claims 4-5 with the target nucleic acid in a cell.

12. A kit characterized in that, The kit comprising the Cas protein of claim 1, the fusion protein of claim 2, or the polynucleotide of claim 3, or the CRISPR-Cas composition of any one of claims 4-5, or the host cell of claim 7, wherein the components of the kit are in the same or different containers; Optionally, the kit comprises one or more buffers.

13. The kit of claim 12, wherein The kit is used for gene or genome editing, disease treatment, targeting a target gene, cleaving one or more of a gene of interest or a non-interest gene.

14. A method of detecting a target nucleic acid in a sample, characterized by, The method comprises contacting a sample with the CRISPR-Cas composition of any one of claims 4-5 and a non-target sequence; detecting a detectable signal generated by cleavage of the non-target sequence, thereby detecting the target nucleic acid; the non-target sequence does not hybridize with the guide RNA.

15. Use of the Cas protein of claim 1, the fusion protein of claim 2, or the polynucleotide of claim 3, or the CRISPR-Cas composition of any one of claims 4-5, or the delivery system of claim 6, or the host cell of claim 7, or the method of claim 8 for targeting and editing a target nucleic acid, or the method of claim 9 for non-specific degradation or cleavage of a single-stranded nucleic acid upon recognition of a target nucleic acid, or the method of claim 10 for targeting and cleaving a double-stranded target nucleic acid in nucleic acid detection.

16. A pharmaceutical composition, characterized by, The kit comprising the CRISPR-Cas composition of any one of claims 4-5, the delivery system of claim 6, or the host cell of claim 7; and a pharmaceutically acceptable excipient; Optionally, the pharmaceutical composition is an injection.

17. A method for diagnosing, preventing or treating a condition or disease in a subject in need thereof, characterized in that, The method comprises administering to a subject an effective amount of the Cas protein of claim 1, the fusion protein of claim 2, or the polynucleotide of claim 3, or the CRISPR-Cas composition of any one of claims 4-5, or the delivery system of claim 6, or the host cell of claim 7, or the pharmaceutical composition of claim 16; Optionally, the condition or disease comprises a cancer or an infectious disease; Optionally, the cancer comprises one or more of Wilms' tumor, Ewing's sarcoma, neuroendocrine tumor, glioblastoma, neuroblastoma, melanoma, skin cancer, breast cancer, colon cancer, rectal cancer, prostate cancer, liver cancer, kidney cancer, pancreatic cancer, lung cancer, biliary tract cancer, cervical cancer, endometrial cancer, esophageal cancer, stomach cancer, head and neck cancer, medullary thyroid cancer, ovarian cancer, glioma, lymphoma, leukemia, myeloma, acute lymphoblastic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, or urinary bladder cancer. Optionally, the condition or disease comprises one or more of cystic fibrosis, Duchenne muscular dystrophy, Becker muscular dystrophy, alpha-1-antitrypsin deficiency, Pompe disease, myotonic muscular dystrophy, Huntington’s disease, fragile X syndrome, Friedreich’s ataxia, amyotrophic lateral sclerosis, frontotemporal dementia, hereditary chronic kidney disease, hyperlipidemia, hypercholesterolemia, Leber’s congenital amaurosis, sickle cell disease, hypercholesterolemia, atherosclerosis, primary hyperoxaluria (PH1), hereditary angioedema (HAE), transthyretin amyloidosis, or beta thalassemia; Optionally, the infectious agent of the infectious disease comprises one or more of human immunodeficiency virus, herpes simplex virus-1, Hepatitis B Virus, or herpes simplex virus-2.

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