CN116200369B

CN116200369B - Cas enzyme and application thereof

Info

Publication number: CN116200369B
Application number: CN202310266207.2A
Authority: CN
Inventors: 李珊珊; 刘锐恒
Original assignee: Shandong Shunfeng Biotechnology Co Ltd
Current assignee: Shandong Shunfeng Biotechnology Co Ltd
Priority date: 2022-03-24
Filing date: 2023-03-15
Publication date: 2024-11-29
Anticipated expiration: 2043-03-15
Also published as: CN116200369A; CN119391671A

Abstract

The invention belongs to the field of nucleic acid editing, in particular to the technical field of regularly clustered interval short palindromic repeat (CRISPR). Specifically, the invention provides a novel Cas enzyme, which belongs to a novel Cas protein and has wide application prospect.

Description

Cas enzyme and application thereof

Technical Field

The invention relates to the field of gene editing, in particular to the technical field of regular clustered interval short palindromic repeat (CRISPR). Specifically, the invention screens out a class of Cas enzyme, and develops a corresponding gene editing tool and application thereof based on the Cas enzyme.

Background

CRISPR/Cas technology is a widely used gene editing technology that uses RNA-guided specific binding of target sequences on the genome and cleavage of DNA to create double strand breaks, site-directed gene editing using biological non-homologous end joining or homologous recombination.

The CRISPR/Cas9 system is the most commonly used type II CRISPR system that recognizes the PAM motif of 3' -NGG and blunt-ends the target sequence. The CRISPR/Cas Type V system is a newly discovered class of CRISPR systems that have a 5' -TTN motif that performs cohesive end cleavage of a target sequence, e.g., cpf1, C2C1, casX, casY. However, the different CRISPR/Cas currently in existence each have different advantages and disadvantages. For example, cas9, C2C1 and CasX each require two RNAs for guide RNAs, whereas Cpf1 requires only one guide RNA and can be used for multiplex gene editing. CasX has a size of 980 amino acids, whereas common Cas9, C2C1, casY and Cpf1 are typically around 1300 amino acids in size. In addition, PAM sequences of Cas9, cpf1, casX, casY are all relatively complex and diverse, while C2C1 recognizes the stringent 5' -TTN, so its target site is easily predicted compared to other systems, thereby reducing potential off-target effects.

In summary, given that the currently available CRISPR/Cas systems are limited by several drawbacks, developing a more robust CRISPR/Cas system with versatile good performance is of great importance for the development of biotechnology.

Disclosure of Invention

The inventors of the present application have unexpectedly found an endonuclease (Cas enzyme) through a lot of experiments and repeated fumbling. Based on this finding, the present inventors developed a new CRISPR/Cas system and a gene editing method and a nucleic acid detection method based on the same.

Cas effector proteins

In one aspect, the invention provides a class of Cas proteins, which are effector proteins in the CRISPR/Cas system, referred to herein as CasN proteins.

The CasN protein disclosed by the invention has lower sequence consistency with the reported Cas protein, and the Cas enzymes are found to belong to an independent evolution branch remarkably and are far away from other proteins by constructing the evolution tree with the Cas enzymes of other types, so that the novel Cas protein belongs to branches of the novel Cas protein.

In one aspect, the invention provides a CasN protein, the CasN protein comprising one or both of the following conserved domains:

The amino acid of the first protective domain is X1NX2X3X4EX5X6X7X8X9X10, wherein ,X1＝D/K/N/P,X2＝F/L/Y,X3＝A/G/L/P/Q/V,X4＝D/F/H/Y,X5＝I/L/T/V,X6＝A/V,X7＝H/K/N/R/T/V/W,X8＝A/Q/R,X9＝I/L/M,X10＝I/L/V;

The amino acids of the second conserved domain are X11X12X13X14X15EX16X17, wherein x11=a/M/Y, x12=f/I/L/M/S/V/Y, x13=i/V, x14=a/C/G, x15=i/L/V, x16=d/H/N/R/Y, x17=a/G/T.

In one embodiment, the amino acid of the first protective domain is X1NX2X3X4EX5X6X7X8X9X10, wherein X1 = D/N/P, X2 = Y, X3 = A/V, X4 = D/F/Y, X5 = T/V, X6 = A/V, X7 = H/K/T, X8 = A, X9 = I/L/M, X10 = I/V.

In one embodiment, the amino acid of the second conserved domain is x11=ajm, x12=i/L/M/V, x13=i, x14=g, x15=i/L, x16=d/H/R, x17=a/G/T, wherein the amino acid of the second conserved domain is x11×12x13x15x15ex 16X 17.

The CasN protein in the invention comprises CasN-1, casN-3, casN-4, casN-6 and CasN-7, and the amino acid sequences of the CasN protein are respectively shown as SEQ ID No. 1-5.

In some embodiments, the amino acid sequence of the CasN protein has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity compared to any of the amino acid sequences of SEQ ID nos. 1-5, and substantially retains the biological function of the sequence from which it is derived.

In one embodiment, the CasN protein amino acid sequence has a sequence with one or more substitutions, deletions or additions of amino acids compared to any of SEQ ID No.1-5, and substantially retains the biological function of the sequence from which it originates, including 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions.

In some embodiments, the CasN proteins of the present invention are capable of recognizing a pre-spacer adjacent motif (PAM), and the target nucleic acid comprises or consists of PAM.

It will be apparent to those skilled in the art that the structure of a protein may be altered without adversely affecting its activity and functionality, for example, one or more conservative amino acid substitutions may be introduced into the amino acid sequence of the protein without adversely affecting the activity and/or three-dimensional structure of the protein molecule. Examples and embodiments of conservative amino acid substitutions are apparent to those skilled in the art. In particular, the amino acid residue may be substituted with another amino acid residue belonging to the same group as the site to be substituted, i.e., with a nonpolar amino acid residue, with a polar uncharged amino acid residue, with a basic amino acid residue, with an acidic amino acid residue. Such substituted amino acid residues may or may not be encoded by the genetic code. Conservative substitutions of one amino acid by another belonging to the same group are within the scope of the invention as long as the substitution does not result in inactivation of the biological activity of the protein. Thus, the proteins of the invention may comprise one or more conservative substitutions in the amino acid sequence, which are preferably made according to table 1. In addition, proteins that also contain one or more other non-conservative substitutions are also contemplated by the present invention, provided that the non-conservative substitutions do not significantly affect the desired function and biological activity of the proteins of the present invention.

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

TABLE 1

Initial residues	Representative substitution	Preferred substitution
			Ala(A)	Val;Leu;Ile	Val
Arg(R)	Lys;Gln;Asn	Lys
			Asn(N)	Gln;His;Lys;Arg	Gln
Asp(D)	Glu	Glu
			Cys(C)	Ser	Ser
Gln(Q)	Asn	Asn
			Glu(E)	Asp	Asp
Gly(G)	Pro;Ala	Ala
			His(H)	Asn;Gln;Lys;Arg	Arg
Ile(I)	Leu;Val;Met;Ala;Phe	Leu
			Leu(L)	Ile;Val;Met;Ala;Phe	Ile
Lys(K)	Arg;Gln;Asn	Arg
			Met(M)	Leu;Phe;Ile	Leu
Phe(F)	Leu;Val;Ile;Ala;Tyr	Leu
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Ser	Ser
Trp(W)	Tyr;Phe	Tyr
			Tyr(Y)	Trp;Phe;Thr;Ser	Phe
Val(V)	Ile;Leu;Met;Phe;Ala	Leu

It is well known in the art that one or more amino acid residues may 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 that have one or more amino acid residues altered from the N and/or C terminus of the Cas protein while retaining their desired functional activity are also within the scope of the invention. These changes may include changes introduced by modern molecular methods such as PCR, including PCR amplification that alters or extends the protein coding sequence by including an amino acid coding sequence in the oligonucleotides used in the PCR amplification.

It will be appreciated that proteins may be altered in a variety of ways, including amino acid substitutions, deletions, truncations and insertions, and that methods for such manipulation are generally known in the art. For example, amino acid sequence variants of the above proteins can be prepared by mutation of DNA. Single or multiple amino acid substitutions, deletions and/or insertions may also be made by other forms of mutagenesis and/or by directed evolution, e.g., using known mutagenesis, recombination and/or shuffling (shuffling) methods, in combination with related screening methods.

Those skilled in the art will appreciate that these minor amino acid changes in the Cas proteins of the invention may occur (e.g., naturally occurring mutations) or be generated (e.g., using r-DNA technology) without loss of protein function or activity. If these mutations occur in the catalytic domain, active site or other functional domain of the protein, the nature of the polypeptide may be altered, but the polypeptide may retain its activity. Smaller effects can be expected if mutations are present that are not close to the catalytic domain, active site or other functional domain.

The skilled artisan can identify the essential amino acids of the Cas protein of the invention according to methods known in the art, such as site-directed mutagenesis or protein evolution or analysis of bioinformatics. The catalytic, active or other functional domains of a protein can also be determined by physical analysis of the structure, such as by techniques such as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, in combination with putative mutations of amino acids at key sites.

In one embodiment, the Cas protein contains the amino acid sequence set forth in any one of SEQ ID nos. 1-5.

In one embodiment, the Cas protein is the amino acid sequence set forth in any one of SEQ ID nos. 1-5.

In one embodiment, the Cas protein is a derivatized protein having the same biological function as a protein having the sequence set forth in any one of SEQ ID nos. 1-5.

The biological functions include, but are not limited to, activity of binding to a guide RNA, endonuclease activity, activity of binding to and cleaving at a specific site of a target sequence under the guidance of a guide RNA, including, but not limited to, cis cleavage activity and/or Trans cleavage activity.

The invention also provides a fusion protein comprising a Cas protein as described above and other modifying moieties.

In one embodiment, the modifying moiety is selected from the group consisting of an additional protein or polypeptide, a detectable label, or any combination thereof.

In one embodiment, the modification moiety is selected from the group consisting of an epitope tag, a reporter gene sequence, a Nuclear Localization Signal (NLS) sequence, a targeting moiety, a transcriptional activation domain (e.g., VP 64), a transcriptional repression domain (e.g., KRAB domain or SID domain), a nuclease domain (e.g., fok 1), and a domain having an activity selected from the group consisting of nucleotide deaminase, methylase activity, demethylase, transcriptional activation activity, transcriptional repression activity, transcriptional release factor activity, histone modification activity, nuclease activity, single-stranded RNA cleavage activity, double-stranded RNA cleavage activity, single-stranded DNA cleavage activity, double-stranded DNA cleavage activity, and nucleic acid binding activity, and any combination thereof. The NLS sequences are well known to those skilled in the art, examples of which include, but are not limited to, the SV40 large T antigen, EGL-13, c-Myc, and TUS proteins.

In one embodiment, the NLS sequence is located at, near, or near the terminus (e.g., N-terminus, C-terminus, or both) of the Cas protein of the invention.

Such epitope tags (tags) are well known to those skilled in the art, including but not limited to His, V5, FLAG, HA, myc, VSV-G, trx, etc., and other suitable epitope tags (e.g., purification, detection, or labeling) may be selected by those skilled in the art.

Such reporter sequences are well known to those skilled in the art, examples of which include, but are not limited to GST, HRP, CAT, GFP, hcRed, dsRed, CFP, YFP, BFP, etc.

In one embodiment, the fusion proteins of the invention comprise a domain capable of binding to a DNA molecule or an intracellular molecule, such as Maltose Binding Protein (MBP), the DNA binding domain of Lex a (DBD), the DBD of GAL4, and the like.

In one embodiment, the fusion proteins of the invention comprise a detectable label, such as a fluorescent dye, e.g., FITC or DAPI.

In one embodiment, the Cas protein of the invention is coupled, conjugated or fused to the modifying moiety, optionally through a linker.

In one embodiment, the modification is directly linked to the N-terminus or C-terminus of the Cas protein of the invention.

In one embodiment, the modifying moiety is linked to the N-terminus or C-terminus of the Cas protein of the invention by a linker. Such linkers are well known in the art, examples of which include, but are not limited to, linkers comprising one or more (e.g., 1,2, 3, 4, or 5) amino acids (e.g., glu or Ser) or amino acid derivatives (e.g., ahx, β -Ala, GABA, or Ava), or PEG, etc.

The Cas protein, protein derivative or fusion protein of the present invention is not limited by the manner of its production, and for example, it may be produced by genetic engineering methods (recombinant techniques) or by chemical synthesis methods.

Nucleic acid of Cas protein

In another aspect, the invention provides an isolated polynucleotide comprising:

(a) Polynucleotide sequences encoding Cas proteins or fusion proteins of the invention;

(b) A polynucleotide having a sequence as shown in any one of SEQ ID No. 6-10;

(c) A sequence having a substitution, deletion, or addition of one or more bases (e.g., a substitution, deletion, or addition of 1, 2,3,4,5, 6, 7, 8, 9, or 10 bases) as compared to the sequence set forth in any one of SEQ ID Nos. 6-10;

(d) The nucleotide sequence has 80% or more homology (preferably 90% or more, more preferably 95% or more, most preferably 98% or more) with any one of the sequences shown in SEQ ID No.6-10, and encodes a polynucleotide of any one of the polypeptides shown in SEQ ID No.1-5, or

(E) A polynucleotide complementary to the polynucleotide of any one of (a) - (d).

In one embodiment, the nucleotide sequence set forth in any one of (a) - (e) is codon optimized for expression in a prokaryotic cell. In one embodiment, the nucleotide sequence set forth in any one of (a) - (e) is codon optimized for expression in a eukaryotic cell.

In one embodiment, the polynucleotide is preferably single-stranded or double-stranded.

Guide RNA (gRNA)

In another aspect, the invention provides a gRNA comprising a framework region, also known as a "protein binding segment" or "protein binding sequence", and a targeting sequence of a targeting nucleic acid, also known as a "targeting segment of a targeting nucleic acid" or a "targeting sequence of a targeting target sequence".

The backbone region of the gRNA is capable of interacting with the Cas protein of the invention, thereby forming a complex of Cas protein and gRNA.

In one embodiment, the gRNA comprises a tracrRNA and a crRNA, wherein the tracrRNA is capable of pairing with a pairing region of the crRNA to form a duplex, and wherein the crRNA further comprises a region that hybridizes to a target sequence (i.e., a targeting sequence of a targeting nucleic acid).

In one embodiment, the sequence of the pairing region of the crRNA and the tracrRNA is shown in any one of SEQ ID Nos. 11 to 16.

In one embodiment, the tracrRNA has the sequence shown in SEQ ID No. 17.

In one embodiment, the gRNA (also known as a guide RNA or guide RNA) of the invention consists of crRNA and tracrRNA molecules that are partially complementary to form a complex, wherein the crRNA comprises a sequence that has sufficient complementarity to a target sequence to hybridize to the complement of the target sequence and direct Cas enzyme binding to the target sequence in a sequence-specific manner. The grnas of the invention include tracrRNA and crRNA.

The targeting sequence of the targeting nucleic acid or targeting segment of the targeting nucleic acid of the invention comprises a nucleotide sequence complementary to a sequence in the target nucleic acid. In other words, the targeting sequence of the targeting nucleic acid or targeting segment of the targeting nucleic acid of the invention interacts with the target nucleic acid in a sequence-specific manner via hybridization (i.e., base pairing). Thus, the targeting sequence of the targeting nucleic acid or targeting segment of the targeting nucleic acid may be altered, or may be modified to hybridize to any desired sequence within the target nucleic acid. The nucleic acid is selected from DNA or RNA.

The targeting sequence of the targeting nucleic acid or the percentage of complementarity between the targeting segment of the targeting nucleic acid and the target sequence of the target nucleic acid can be at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%).

The "framework region", "protein binding segment", "protein binding sequence" of the gRNA of the invention can interact with a CRISPR protein (or Cas protein). The gRNA of the invention directs its interacting Cas protein to a specific nucleotide sequence within the target nucleic acid through the action of the targeting sequence of the targeting nucleic acid.

The gRNA of the invention is capable of forming a complex with the Cas protein.

Carrier body

The invention also provides a vector comprising a Cas protein, an isolated nucleic acid molecule or a polynucleotide as described above, preferably further comprising a regulatory control element operably linked thereto.

In one embodiment, the regulatory element is selected from one or more of the group consisting of an enhancer, a transposon, a promoter, a terminator, a leader sequence, a polyadenylation sequence, and a marker gene.

In one embodiment, the vector comprises a cloning vector, an expression vector, a shuttle vector, an integration vector.

In some embodiments, the vectors included in the system are viral vectors (e.g., retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated vectors, and herpes simplex vectors), but may also be of the plasmid, viral, cosmid, phage, etc. type, which are well known to those skilled in the art.

CRISPR system

The present invention provides an engineered non-naturally occurring vector system, or CRISPR-Cas system, comprising a Cas protein or a nucleic acid sequence encoding the Cas protein and a nucleic acid encoding one or more guide RNAs comprising a region that binds to the Cas protein and a targeting sequence that targets the nucleic acid.

In one embodiment, the nucleic acid sequence encoding the Cas protein and the nucleic acid encoding one or more guide RNAs are synthetic.

In one embodiment, the nucleic acid sequence encoding the Cas protein and the nucleic acid encoding one or more guide RNAs do not co-occur naturally.

The one or more guide RNAs target one or more target sequences in the cell. The one or more target sequences hybridize to a genomic locus of a DNA molecule encoding one or more gene products and the Cas protein is directed to the genomic locus of the DNA molecule of the one or more gene products, and the Cas protein, upon reaching the target sequence position, modifies, edits or cleaves the target sequence, whereby expression of the one or more gene products is altered or modified.

The cells of the invention include one or more of animals, plants or microorganisms.

In some embodiments, the Cas protein is codon optimized for expression in a cell.

In some embodiments, the Cas protein directs cleavage of one or both strands at the target sequence position.

The invention also provides an engineered non-naturally occurring carrier system that can include one or more carriers comprising:

a) A first regulatory element operably linked to a gRNA comprising a region that binds to the Cas protein and a targeting sequence that targets a nucleic acid;

b) A second regulatory element operably linked to the Cas protein;

Wherein components (a) and (b) are on the same or different supports of the system.

The first and second regulatory elements include promoters (e.g., constitutive promoters or inducible promoters), enhancers (e.g., 35S promoter or 35S enhanced promoter), internal Ribosome Entry Sites (IRES), and other expression control elements (e.g., transcriptional termination signals, such as polyadenylation signals and poly U sequences).

In some embodiments, the vectors in the system are viral vectors (e.g., retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated vectors, and herpes simplex vectors), but may also be of the plasmid, viral, cosmid, phage, etc. type, which are well known to those skilled in the art.

In some embodiments, the systems provided herein are in a delivery system. In some embodiments, the delivery system is a nanoparticle, liposome, exosome, microvesicle, or gene gun.

In one embodiment, the target sequence is a DNA or RNA sequence from a prokaryotic or eukaryotic cell. In one embodiment, the target sequence is a non-naturally occurring DNA or RNA sequence.

In one embodiment, the target sequence is present in a cell. In one embodiment, the target sequence is present in the nucleus or in the cytoplasm (e.g., organelle). In one embodiment, the cell is a eukaryotic cell. In other embodiments, the cell is a prokaryotic cell.

In one embodiment, the Cas protein has one or more NLS sequences attached. In one embodiment, the fusion protein comprises one or more NLS sequences. In one embodiment, the NLS sequence is linked to the N-terminus or C-terminus of the protein. In one embodiment, the NLS sequence is fused to the N-terminus or C-terminus of the protein.

In another aspect, the invention relates to an engineered CRISPR system comprising the Cas protein described above and one or more guide RNAs, wherein the guide RNAs comprise a region that binds to the Cas protein and a targeting sequence that targets a nucleic acid, the Cas protein being capable of binding to the guide RNAs and targeting a target nucleic acid sequence that is complementary to the targeting sequence.

Protein-nucleic acid complexes/compositions

In another aspect, the invention provides a complex or composition comprising:

(i) A protein component selected from the group consisting of the Cas protein, the derivatized protein, or the fusion protein described above, and any combination thereof, and

(Ii) A nucleic acid component selected from a gRNA, or a nucleic acid encoding the gRNA, or a precursor RNA of the gRNA, or a precursor RNA nucleic acid encoding the gRNA, the gRNA comprising a region that binds to the Cas protein, and a targeting sequence for a targeting nucleic acid;

The protein component and the nucleic acid component are bound to each other to form a complex.

In one embodiment, the nucleic acid component is a guide RNA in a CRISPR-Cas system.

In one embodiment, the complex or composition is non-naturally occurring or modified. In one embodiment, at least one component of the complex or composition is non-naturally occurring or modified. In one embodiment, the first component is non-naturally occurring or modified, and/or the second component is non-naturally occurring or modified.

Activated CRISPR complexes

In another aspect, the invention also provides an activated CRISPR complex comprising (1) a protein component selected from the group consisting of the Cas protein, the derivatized protein, or the fusion protein of the invention, and any combination thereof, (2) a nucleic acid component selected from the group consisting of a gRNA, or a nucleic acid encoding the gRNA, or a precursor RNA of the gRNA, or a precursor RNA nucleic acid encoding the gRNA, the gRNA comprising a region that binds to the Cas protein and a targeting sequence that targets the nucleic acid, and (3) a target sequence that binds to the gRNA. Preferably, the binding is binding to the target nucleic acid through a targeting sequence of the targeting nucleic acid on the gRNA.

The term "activated CRISPR complex", "activated complex" or "ternary complex" as used herein refers to a complex in a CRISPR system where Cas protein, gRNA bind to or are modified with a target nucleic acid.

The Cas proteins and grnas of the invention can form binary complexes that are activated when bound to a nucleic acid substrate that is complementary to a targeting sequence in the gRNA (or, referred to as, a guide sequence that hybridizes to a target nucleic acid) to form an activated CRISPR complex. In some embodiments, the targeting sequence of the gRNA is perfectly matched to the target substrate. In other embodiments, the targeting sequence of the gRNA matches a portion (continuous or discontinuous) of the target substrate.

In preferred embodiments, the activated CRISPR complex may exhibit a sidebranch nuclease cleavage activity, which refers to the nonspecific cleavage activity or hackle activity of the activated CRISPR complex on single-stranded nucleic acids, also known in the art as trans cleavage activity.

Delivery and delivery compositions

Cas proteins, grnas, fusion proteins, nucleic acid molecules, vectors, systems, complexes, and compositions of the invention may be delivered by any method known in the art. Such methods include, but are not limited to, electroporation, lipofection, nuclear transfection, microinjection, sonoporation, gene gun, calcium phosphate mediated transfection, cationic transfection, lipofection, dendritic transfection, heat shock transfection, nuclear transfection, magnetic transfection, lipofection, puncture transfection, optical transfection, reagent enhanced nucleic acid uptake, and delivery via liposomes, immunoliposomes, virosomes, artificial virosomes, and the like.

Accordingly, in another aspect, the present invention provides a delivery composition comprising a delivery vehicle, and one or more selected from the group consisting of Cas proteins, fusion proteins, nucleic acid molecules, vectors, systems, complexes, and compositions of the present invention.

In one embodiment, the delivery vehicle is a particle.

In one embodiment, the delivery vehicle is selected from the group consisting of a lipid particle, a sugar particle, a metal particle, a protein particle, a liposome, an exosome, a microbubble, a gene gun, or a viral vector (e.g., replication defective retrovirus, lentivirus, adenovirus, or adeno-associated virus).

Host cells

The invention also relates to an in vitro, ex vivo or in vivo cell or cell line or progeny thereof comprising a Cas protein, a fusion protein, a nucleic acid molecule, a protein-nucleic acid complex, an activated CRISPR complex, a vector, a delivery composition of the invention.

In certain embodiments, the cell is a prokaryotic cell.

In certain embodiments, the cell is a eukaryotic cell. In certain embodiments, the cell is a mammalian cell. In certain embodiments, the cell is a human cell. In certain embodiments, the cell is a non-human mammalian cell, e.g., a non-human primate, bovine, ovine, porcine, canine, simian, rabbit, rodent (e.g., rat or mouse) cell. In certain embodiments, the cells are non-mammalian eukaryotic cells, such as cells of poultry birds (e.g., chickens), fish, or crustaceans (e.g., clams, shrimps). In certain embodiments, the cell is a plant cell, e.g., a cell of a monocot or dicot or a cell of a cultivated plant or a food crop such as tapioca, corn, sorghum, soybean, wheat, oat, or rice, e.g., an algae, tree, or production plant, fruit, or vegetable (e.g., a tree such as citrus, nut, eggplant, cotton, tobacco, tomato, grape, coffee, cocoa, etc.).

In certain embodiments, the cell is a stem cell or stem cell line.

In certain instances, a host cell of the invention comprises a modification of a gene or genome that is not present in its wild type.

Gene editing method and application

The Cas protein, nucleic acid, the composition described above, the CIRSPR/Cas system described above, the vector system described above, the delivery composition described above, or the activated CRISPR complex described above, or the host cell of the present invention may be used for any one or any of several purposes, targeting and/or editing a target nucleic acid, cleaving double-stranded DNA, single-stranded DNA, or single-stranded RNA, non-specifically cleaving and/or degrading a collateral nucleic acid, non-specifically cleaving a single-stranded nucleic acid, nucleic acid detection, detecting a nucleic acid in a target sample, specifically editing a double-stranded nucleic acid, base editing a single-stranded nucleic acid. In other embodiments, it may also be used to prepare reagents or kits for any one or any of several of the uses described above.

The invention also provides the use of the Cas protein, nucleic acid, composition, CIRSPR/Cas system, vector system, delivery composition, or activated CRISPR complex described above in gene editing, gene targeting, or gene cleavage, or in the preparation of a reagent or kit for gene editing, gene targeting, or gene cleavage.

In one embodiment, the gene editing, gene targeting or gene cleaving is performed intracellularly and/or extracellularly.

The invention also provides a method of editing, targeting, or cleaving a target nucleic acid comprising contacting the target nucleic acid with the Cas protein, nucleic acid, composition, CIRSPR/Cas system, vector system, delivery composition, or activated CRISPR complex described above. In one embodiment, the method is editing, targeting, or cleaving a target nucleic acid inside or outside a cell.

The gene editing or editing target nucleic acids include modifying genes, knocking out genes, altering expression of gene products, repairing mutations, and/or inserting polynucleotides, gene mutations.

The editing may be performed in prokaryotic and/or eukaryotic cells.

In another aspect, the invention also provides the use of the Cas protein, the nucleic acid, the composition, the CIRSPR/Cas system, the vector system, the delivery composition, or the activated CRISPR complex described above in nucleic acid detection, or in the preparation of a reagent or kit for nucleic acid detection.

In another aspect, the invention also provides a method of cleaving a single-stranded nucleic acid, the method comprising contacting a nucleic acid population with the Cas protein described above and a gRNA comprising a region that binds to the Cas protein and a targeting sequence that targets a nucleic acid, wherein the nucleic acid population comprises a target nucleic acid and a plurality of non-target single-stranded nucleic acids, the Cas protein cleaving the plurality of non-target single-stranded nucleic acids.

The gRNA is capable of binding to the Cas protein.

The gRNA is capable of targeting the target nucleic acid.

The contacting may be inside a cell in vitro, ex vivo or in vivo.

Preferably, the cleavage of single-stranded nucleic acid is a nonspecific cleavage.

In another aspect, the invention also provides the use of the Cas protein, the nucleic acid, the composition, the CIRSPR/Cas system, the vector system, the delivery composition, or the activated CRISPR complex described above for non-specifically cleaving a single stranded nucleic acid, or for the preparation of a reagent or kit for non-specifically cleaving a single stranded nucleic acid.

In another aspect, the invention also provides a kit for gene editing, gene targeting or gene cleavage comprising the Cas protein, gRNA, nucleic acid, the composition, the CIRSPR/Cas system, the vector system, the delivery composition, the activated CRISPR complex or the host cell described above.

In another aspect, the invention also provides a kit for detecting a target nucleic acid in a sample, the kit comprising (a) a Cas protein, or a nucleic acid encoding the Cas protein, (b) a guide RNA, or a nucleic acid encoding the guide RNA, or a precursor RNA comprising the guide RNA, or a nucleic acid encoding the precursor RNA, and (c) a single-stranded nucleic acid detector that is single-stranded and does not hybridize to the guide RNA.

It is known in the art that precursor RNAs can be cleaved or processed into the mature guide RNAs described above.

In another aspect, the invention provides the use of the Cas protein, the nucleic acid, the composition, the CIRSPR/Cas system, the vector system, the delivery composition, the activated CRISPR complex, or the host cell described above in the preparation of a formulation or kit for:

(i) Gene or genome editing;

(ii) Target nucleic acid detection and/or diagnosis;

(iii) Editing a target sequence in a target locus to modify an organism or a non-human organism;

(iv) Targeting a target gene;

(v) Cutting the target gene.

Preferably, the gene or genome editing is performed in or out of a cell.

Preferably, the target nucleic acid detection and/or diagnosis is performed in vitro.

Preferably, the treatment of the disease is treatment of a condition caused by a defect in the target sequence in the target locus.

In another aspect, the invention provides a method of detecting a target nucleic acid in a sample, the method comprising contacting the sample with the Cas protein, a gRNA (guide RNA) comprising a region that binds to the Cas protein and a targeting sequence that hybridizes to the target nucleic acid, and a single-stranded nucleic acid detector, detecting a detectable signal generated by cleavage of the single-stranded nucleic acid detector by the Cas protein, thereby detecting the target nucleic acid, the single-stranded nucleic acid detector not hybridizing to the gRNA.

Method for specifically modifying target nucleic acid

In another aspect, the invention also provides a method of specifically modifying a target nucleic acid, comprising contacting the target nucleic acid with the Cas protein, the nucleic acid, the composition, the CIRSPR/Cas system, the vector system, the delivery composition, or the activated CRISPR complex.

The specific modification may occur in vivo or in vitro.

The specific modification may occur either intracellularly or extracellularly.

In some cases, the cell is selected from a prokaryotic cell or a eukaryotic cell, e.g., an animal cell, a plant cell, or a microbial cell.

In one embodiment, the modification refers to cleavage of the target sequence, e.g., single/double strand cleavage of DNA, or single strand cleavage of RNA.

In some cases, the method further comprises contacting the target nucleic acid with a donor polynucleotide, wherein the donor polynucleotide, a portion of the donor polynucleotide, a copy of the donor polynucleotide, or a portion of a copy of the donor polynucleotide is integrated into the target nucleic acid.

In one embodiment, the modification further comprises inserting an editing template (e.g., an exogenous nucleic acid) into the break.

In one embodiment, the method further comprises contacting an editing template with the target nucleic acid or delivering into a cell comprising the target nucleic acid. In this embodiment, the method repairs the disrupted target gene by homologous recombination with an exogenous template polynucleotide, in some embodiments the repair results in a mutation, including an insertion, deletion, or substitution of one or more nucleotides of the target gene, in other embodiments the mutation results in one or more amino acid changes in a protein expressed from a gene comprising the target sequence.

Detection (non-specific cleavage)

In another aspect, the invention provides a method of detecting a target nucleic acid in a sample, the method comprising contacting the sample with the Cas protein described above, a nucleic acid, the composition described above, the CIRSPR/Cas system described above, the vector system described above, the delivery composition described above, or the activated CRISPR complex described above, and a single stranded nucleic acid detector, detecting a detectable signal produced by cleavage of the single stranded nucleic acid detector by the Cas protein, thereby detecting the target nucleic acid.

In the present invention, the target nucleic acid includes ribonucleotides or deoxyribonucleotides, and includes single-stranded nucleic acids, double-stranded nucleic acids, such as single-stranded DNA, double-stranded DNA, single-stranded RNA, double-stranded RNA.

In one embodiment, the target nucleic acid is derived from a sample of a virus, bacterium, microorganism, soil, water source, human, animal, plant, or the like. Preferably, the target nucleic acid is a product of enrichment or amplification by PCR, NASBA, RPA, SDA, LAMP, HAD, NEAR, MDA, RCA, LCR, RAM or the like.

In one embodiment, the target nucleic acid is a viral nucleic acid, a bacterial nucleic acid, a specific nucleic acid associated with a disease, such as a specific mutation site or SNP site or a nucleic acid that differs from a control, preferably the virus is a plant virus or an animal virus, e.g., papilloma virus, hepadnavirus, herpesvirus, adenovirus, poxvirus, parvovirus, coronavirus, preferably the virus is a coronavirus, preferably SARS, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, mers-Cov.

In the present invention, the gRNA has a degree of match of at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90% with the target sequence on the target nucleic acid.

In one embodiment, when the target sequence contains one or more characteristic sites (e.g., specific mutation sites or SNPs), the characteristic sites are perfectly matched to the gRNA.

In one embodiment, the detection method may comprise one or more grnas with targeting sequences that are different from each other, targeting different target sequences.

In the present invention, the single-stranded nucleic acid detector includes, but is not limited to, single-stranded DNA, single-stranded RNA, DNA-RNA hybrids, nucleic acid analogs, base modifications, single-stranded nucleic acid detectors containing no base spacers, and the like, "nucleic acid analogs" include, but are not limited to, locked nucleic acids, bridged nucleic acids, morpholino nucleic acids, ethylene glycol nucleic acids, hexitol nucleic acids, threose nucleic acids, arabinose nucleic acids, 2' oxymethyl RNA, 2' methoxyacetyl RNA, 2' fluoro RNA, 2' amino RNA, 4' thio RNA, and combinations thereof, including optional ribonucleotides or deoxyribonucleotide residues.

In the present invention, the detectable signal is achieved by visual based detection, sensor based detection, color detection, fluorescent signal based detection, gold nanoparticle based detection, fluorescence polarization, colloidal phase change/dispersion, electrochemical detection, and semiconductor based detection.

In the present invention, it is preferable that both ends of the single-stranded nucleic acid detector are provided with a fluorescent group and a quenching group, respectively, and that the single-stranded nucleic acid detector may exhibit a detectable fluorescent signal when cleaved. The fluorescent group is one or more selected from FAM, FITC, VIC, JOE, TET, CY, CY5, ROX, texas Red or LC RED460, and the quenching group is one or more selected from BHQ1, BHQ2, BHQ3, dabcy1 or Tamra.

In other embodiments, the 5 'end and the 3' end of the single-stranded nucleic acid detector are respectively provided with different labeling molecules, and colloidal gold test results before the single-stranded nucleic acid detector is cut by the Cas protein and after the single-stranded nucleic acid detector is cut by the Cas protein are detected by a colloidal gold detection method, and the single-stranded nucleic acid detector shows different color development results on a colloidal gold detection line and a quality control line before the single-stranded nucleic acid detector is cut by the Cas protein and after the single-stranded nucleic acid detector is cut by the Cas protein.

In some embodiments, the method of detecting a target nucleic acid may further comprise comparing the level of the detectable signal to a reference signal level, and determining the amount of the target nucleic acid in the sample based on the level of the detectable signal.

In some embodiments, the method of detecting a target nucleic acid may further comprise using RNA reporter nucleic acid and DNA reporter nucleic acid (e.g., fluorescent colors) on different channels, and sampling based on combining (e.g., using a minimum or product) the levels of the detectable signals by measuring the signal levels of the RNA and DNA reporter molecules, and determining the levels of the detectable signals by measuring the amounts of target nucleic acid in the RNA and DNA reporter molecules.

In one embodiment, the target gene is present in a cell.

In one embodiment, the cell is a prokaryotic cell.

In one embodiment, the cell is a eukaryotic cell.

In one embodiment, the cell is an animal cell.

In one embodiment, the cell is a human cell.

In one embodiment, the cell is a plant cell, such as a cell of a cultivated plant (e.g., cassava, maize, sorghum, wheat, or rice), algae, tree, or vegetable.

In one embodiment, the target gene is present in an in vitro nucleic acid molecule (e.g., a plasmid).

In one embodiment, the target gene is present in a plasmid.

Definition of terms

In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Further, the procedures of molecular genetics, nucleic acid chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics and recombinant DNA, etc., as used herein, are all conventional procedures widely used in the corresponding field. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.

In the present invention, amino acid residues may be represented by single letters or three letters, such as alanine (Ala, A), valine (Val, V), glycine (Gly, G), leucine (Leu, L), glutamine (Gln, Q), phenylalanine (Phe, F), tryptophan (Trp, W), tyrosine (Tyr, Y), aspartic acid (Asp, D), asparagine (Asn, N), glutamic acid (Glu, E), lysine (Lys, K), methionine (Met, M), serine (Ser, S), threonine (Thr, T), cysteine (Cys, C), proline (Pro, P), isoleucine (Ile, I), histidine (His, H), arginine (Arg, R).

Cas proteins

In the present invention, cas protein, cas enzyme, cas effect protein are used interchangeably, and the present inventors have found and identified for the first time a Cas effect protein having an amino acid sequence selected from the group consisting of:

(i) Any one of SEQ ID No. 1-5;

(ii) A sequence having one or more amino acid substitutions, deletions or additions (e.g.1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions) compared to the sequence shown in any of SEQ ID Nos. 1-5, or

(Iii) A sequence having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence set forth in any one of SEQ ID nos. 1-5.

Nucleic acid cleavage or cleavage nucleic acids herein include DNA or RNA cleavage (Cis cleavage), cleavage of DNA or RNA in a side branch nucleic acid substrate (single-stranded nucleic acid substrate) in a target nucleic acid produced by a Cas enzyme described herein (i.e., non-specific or non-targeted, trans cleavage). In some embodiments, the cleavage is a double-stranded DNA break. In some embodiments, the cleavage is a single-stranded DNA cleavage or a single-stranded RNA cleavage.

CRISPR system

As used herein, the term "regularly clustered, spaced short palindromic repeats (CRISPR) -CRISPR-associated (Cas) (CRISPR-Cas) system" or "CRISPR system" is used interchangeably and has the meaning commonly understood by those skilled in the art, which generally comprises transcripts or other elements related to the expression of a CRISPR-associated ("Cas") gene, or transcripts or other elements capable of directing the activity of the Cas gene.

CRISPR/Cas complexes

As used herein, the term "CRISPR/Cas complex" refers to a complex formed by the binding of a guide RNA (guide RNA) to a Cas protein, comprising a backbone region that hybridizes to a targeting sequence of a target sequence and binds to a Cas protein, which complex is capable of recognizing and cleaving a polynucleotide that is capable of hybridizing to the guide RNA.

Guide RNA (guide RNA, gRNA)

As used herein, the terms "guide RNA", "guide sequence" are used interchangeably and have the meaning commonly understood by those skilled in the art. In general, the guide RNA can comprise, consist essentially of, or consist of a backbone region and a targeting sequence of a targeting nucleic acid.

In certain instances, a targeting sequence 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 targeting sequence and its corresponding target sequence is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% when optimally aligned. It is within the ability of one of ordinary skill in the art to determine the optimal alignment. For example, there are published and commercially available alignment algorithms and programs such as, but not limited to, the Smith-Waterman algorithm (Smith-Waterman), bowtie, geneious, biopython, and SeqMan in ClustalW, matlab.

Target sequence

"Target sequence" refers to a polynucleotide targeted by a targeting sequence in a gRNA, e.g., a sequence that has complementarity to the targeting sequence, wherein hybridization between the target sequence and the targeting sequence will promote the formation of a CRISPR/Cas complex (including Cas proteins and grnas). Complete complementarity is not necessary so long as sufficient complementarity exists to cause hybridization and promote the formation of a CRISPR/Cas complex.

The target sequence may comprise any polynucleotide, such as DNA or RNA. In some cases, the target sequence is located either inside or outside the cell. In some cases, the target sequence is located in the nucleus or cytoplasm of the cell. In some cases, the target sequence may be located within an organelle of a eukaryotic cell, such as a mitochondria or chloroplast. Sequences or templates that can be used for recombination into a target locus comprising the target sequence are referred to as "editing templates" or "editing polynucleotides" or "editing sequences". In one embodiment, the editing template is an exogenous nucleic acid. In one embodiment, the recombination is homologous recombination.

In the present invention, a "target sequence" or "target polynucleotide" or "target nucleic acid" may be any polynucleotide that is endogenous or exogenous to a cell (e.g., a eukaryotic cell). For example, the target polynucleotide may be a polynucleotide that is present in the nucleus of a eukaryotic cell. The target polynucleotide may be a sequence encoding a gene product (e.g., a protein) or a non-coding sequence (e.g., a regulatory polynucleotide or unwanted DNA). In some cases, the target sequence should be related to the Protospacer Adjacent Motif (PAM).

Single-stranded nucleic acid detector

The single-stranded nucleic acid detector according to the present invention means a detector comprising a sequence of 2 to 200 nucleotides, preferably 2 to 150 nucleotides, preferably 3 to 100 nucleotides, preferably 3 to 30 nucleotides, preferably 4 to 20 nucleotides, more preferably 5 to 15 nucleotides. Preferably a single-stranded DNA molecule, a single-stranded RNA molecule or a single-stranded DNA-RNA hybrid.

The single-stranded nucleic acid detector comprises different reporter groups or marker molecules at both ends, which do not exhibit a reporter signal when in an initial state (i.e., not cleaved), and which exhibit a detectable signal when cleaved, i.e., a detectable distinction between cleaved and pre-cleaved.

In one embodiment, the reporter or marker molecule comprises a fluorescent group and a quenching group, wherein the fluorescent group is selected from one or more of FAM, FITC, VIC, JOE, TET, CY, CY5, ROX, texas Red or LC RED460, and the quenching group is selected from one or more of BHQ1, BHQ2, BHQ3, dabcy1 or Tamra.

In one embodiment, the single stranded nucleic acid detector has a first molecule (e.g., FAM or FITC) attached to the 5 'end and a second molecule (e.g., biotin) attached to the 3' end. The reaction system containing the single-stranded nucleic acid detector is matched with a flow strip to detect target nucleic acid (preferably, a colloidal gold detection mode). The flow strip is designed with two capture lines, with an antibody binding to a first molecule (i.e., a first molecular antibody) at the sample contact end (colloidal gold), an antibody binding to the first molecular antibody at the first line (control line), and an antibody binding to a second molecule (i.e., a second molecular antibody, such as avidin) at the second line (test line). When the reaction flows along the strip, the first molecular antibody binds to the first molecule carrying the cleaved or uncleaved oligonucleotide to the capture line, the cleaved reporter will bind to the antibody of the first molecular antibody at the first capture line, and the uncleaved reporter will bind to the second molecular antibody at the second capture line. Binding of the reporter group at each line will result in a strong readout/signal (e.g., color). As more reporter is cut, more signal will accumulate at the first capture line and less signal will appear at the second line. In certain aspects, the invention relates to the use of a flow strip as described herein for detecting nucleic acids. In certain aspects, the invention relates to a method of detecting nucleic acids using a flow strip as defined herein, e.g. a (lateral) flow test or a (lateral) flow immunochromatographic assay. In certain aspects, the molecules in the single stranded nucleic acid detector may be interchanged or the positions of the molecules may be changed, so long as the reporting principle is the same or similar to that of the present invention, and the modified manner is also included in the present invention.

The detection method provided by the invention can be used for quantitative detection of target nucleic acid to be detected. The quantitative detection index can be quantified according to the signal intensity of the reporter group, such as the luminous intensity of the fluorescent group, the width of the color-developing strip, and the like.

Wild type

As used herein, the term "wild-type" has the meaning commonly understood by those skilled in the art, which refers to a typical form of an organism, strain, gene, or a characteristic that, when it exists in nature, differs from a mutant or variant form, which may be isolated from a source in nature and not intentionally modified by man.

Derivatization

As used herein, the term "derivatization" refers to a chemical modification of an amino acid, polypeptide, or protein in which one or more substituents have been covalently attached to the amino acid, polypeptide, or protein. Substituents may also be referred to as side chains.

A derivatized protein is a derivative of the protein, in general, derivatization of the protein does not adversely affect the desired activity of the protein (e.g., binding to a guide RNA, endonuclease activity, binding to a specific site of a target sequence under the guidance of a guide RNA and cleavage activity), that is, the derivative of the protein has the same activity as the protein.

Derivatizing proteins

Also referred to as "protein derivatives" refers to modified forms of a protein, for example, wherein one or more amino acids of the protein may be deleted, inserted, modified and/or substituted.

Non-naturally occurring

As used herein, the terms "non-naturally occurring" or "engineered" are used interchangeably and refer to human involvement. When these terms are used to describe a nucleic acid molecule or polypeptide, it means that the nucleic acid molecule or polypeptide is at least substantially free from at least one other component to which it is associated in nature or as found in nature.

Orthologs (orthologue, ortholog)

As used herein, the term "ortholog (orthologue, ortholog)" has the meaning commonly understood by those skilled in the art. As a further guidance, an "ortholog" of a protein as described herein refers to a protein belonging to a different species that performs the same or similar function as the protein as its ortholog.

Identity of

As used herein, the term "identity" is used to refer to the match of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matched positions shared by the two sequences divided by the number of positions to be compared x 100. For example, if 6 out of 10 positions of two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 positions in total are matched). Typically, the comparison is made when two sequences are aligned to produce maximum identity. Such alignment may be achieved using, for example, the method of Needleman et al (1970) J.mol.biol.48:443-453, which may be conveniently performed by a computer program such as the Align program (DNAstar, inc.). The percent identity between two amino acid sequences can also be determined using the algorithm of E.Meyers and W.Miller (Comput. Appl biosci.,4:11-17 (1988)) which has been integrated into the ALIGN program (version 2.0), using the PAM120 weight residue table (weight residue table), the gap length penalty of 12 and the gap penalty of 4. Furthermore, percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J MoI biol.48:444-453 (1970)) algorithms that have been incorporated into the GAP program of the GCG software package (available on www.gcg.com) using the Blossum 62 matrix or PAM250 matrix and the GAP weights (GAP WEIGHT) of 16, 14, 12, 10, 8, 6 or 4 and the length weights of 1,2, 3, 4, 5 or 6.

Carrier body

The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid molecule linked thereto. Vectors include, but are not limited to, single-stranded, double-stranded, or partially double-stranded nucleic acid molecules, nucleic acid molecules comprising one or more free ends, no free ends (e.g., circular), nucleic acid molecules comprising DNA, RNA, or both, and other diverse polynucleotides known in the art. The vector may be introduced into a host cell by transformation, transduction or transfection such that the genetic material elements carried thereby are expressed in the host cell. A vector may be introduced into a host cell to thereby produce a transcript, protein, or peptide, including from a protein, fusion protein, isolated nucleic acid molecule, or the like (e.g., a CRISPR transcript, such as a nucleic acid transcript, protein, or enzyme) as described herein. A vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain a replication origin.

One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA fragments may be inserted, for example, by standard molecular cloning techniques.

Another type of vector is a viral vector in which a virus-derived DNA or RNA sequence is present in a vector used to package a virus (e.g., retrovirus, replication-defective retrovirus, adenovirus, replication-defective adenovirus, and adeno-associated virus). Viral vectors also comprise polynucleotides carried by a virus for transfection into a host cell. Certain vectors (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors) are capable of autonomous replication in a host cell into which they are introduced.

Other vectors (e.g., 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 the expression of genes to which they are operably linked. Such vectors are referred to herein as "expression vectors".

Host cells

As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including, but not limited to, prokaryotic cells such as e.g. escherichia coli or bacillus subtilis, eukaryotic cells such as microbial cells, fungal cells, animal cells and plant cells.

Those skilled in the art will appreciate that the design of the expression vector may depend on factors such as the choice of host cell to be transformed, the desired level of expression, and the like.

Regulatory element

As used herein, the term "regulatory element" is intended to include promoters, enhancers, internal Ribosome Entry Sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly U sequences), a detailed description of which may be found in Goeddel, gene expression techniques: enzymatic methods (GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY) 185, academic Press (ACADEMIC PRESS), san Diego, calif., 1990. In some cases, regulatory elements include those sequences that direct constitutive expression of a nucleotide sequence in many types of host cells as well as those sequences that direct expression of the nucleotide sequence in only certain host cells (e.g., tissue-specific regulatory sequences). Tissue-specific promoters may primarily direct expression in a desired tissue of interest, such as muscle, neurons, bone, skin, blood, specific organs (e.g., liver, pancreas), or specific cell types (e.g., lymphocytes). In some cases, regulatory elements may also direct expression in a time-dependent manner (e.g., in a cell cycle-dependent or developmental stage-dependent manner), which may or may not be tissue or cell type specific. In some cases, the term "regulatory element" encompasses enhancer elements such as WPRE, CMV enhancer, R-U5' fragment in the LTR of HTLV-I ((mol. CCell. Biol., vol. 8 (1), pp. 466-472, 1988)), SV40 enhancer, and intron sequences between exons 2 and 3 of rabbit beta-globin (Proc. Natl. Acad. Sci. USA., vol. 78 (3), pp. 1527-31, 1981).

Promoters

As used herein, the term "promoter" has a meaning well known to those skilled in the art and refers to a non-coding nucleotide sequence located upstream of a gene that is capable of initiating expression of a downstream gene. A constitutive (constitutive) promoter is a nucleotide sequence which, when operably linked to a polynucleotide encoding or defining a gene product, results in the production of the gene product in a cell under most or all physiological conditions of the cell. An inducible promoter is a nucleotide sequence which, when operably linked to a polynucleotide encoding or defining a gene product, results in the production of the gene product in a cell, essentially only when an inducer corresponding to the promoter is present in the cell. A tissue-specific promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, results in the production of the gene product in a cell, essentially only if the cell is a cell of the tissue type to which the promoter corresponds.

NLS

A "nuclear localization signal" or "nuclear localization sequence" (NLS) is an amino acid sequence that "tags" a protein for introduction into the nucleus by nuclear transport, i.e., a protein with NLS is transported to the nucleus. Typically, NLS contains positively charged Lys or Arg residues exposed at the protein surface. Exemplary nuclear localization sequences include, but are not limited to, NLS from SV40 large T antigen, EGL-13, c-Myc, and TUS proteins. In some embodiments, the NLS comprises PKKKRKV sequence. In some embodiments, the NLS comprises the AVKRPAATKKAGQAKKKKLD sequence. In some embodiments, the NLS comprises the PAAKRVKLD sequence. In some embodiments, the NLS comprises the MSRRRKANPTKLSENAKKLAKEVEN sequence. In some embodiments, the NLS comprises the KLKIKRPVK sequence. Other nuclear localization sequences include, but are not limited to, the acidic M9 domain of hnRNP A1, sequences KIPIK in yeast transcription repressor Mat. Alpha.2, and PY-NLS.

Operatively connected to

As used herein, the term "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the one or more regulatory elements 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).

Complementarity and method of detecting complementary

As used herein, the term "complementarity" refers to the ability of a nucleic acid to form one or more hydrogen bonds with another nucleic acid sequence by means of a conventional watson-crick or other non-conventional type. Percent complementarity means 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 of 10 are 50%, 60%, 70%, 80%, 90%, and 100% complementary). "fully complementary" means that all consecutive residues of one nucleic acid sequence form hydrogen bonds with the same number of consecutive residues in one second nucleic acid sequence. "substantially complementary" as used herein refers to a degree of complementarity of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% over 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 to two nucleic acids that hybridize under stringent conditions.

Stringent conditions

As used herein, "stringent conditions" for hybridization refers to conditions under which a nucleic acid having complementarity to a target sequence hybridizes predominantly to the target sequence and does not substantially hybridize to non-target sequences. Stringent conditions are typically sequence-dependent and will vary depending on a number of factors. In general, the longer the sequence, the higher the temperature at which the sequence specifically hybridizes to its target sequence.

Hybridization

The term "hybridization" or "complementary" or "substantially complementary" means that a nucleic acid (e.g., RNA, DNA) comprises a nucleotide sequence that enables it to bind non-covalently, i.e., form base pairs and/or G/U base pairs with another nucleic acid in a sequence-specific, antiparallel manner (i.e., the nucleic acid specifically binds to the complementary nucleic acid), "anneal" or "hybridize".

Hybridization requires that the two nucleic acids contain complementary sequences, although there may be mismatches between bases. Suitable conditions for hybridization between two nucleic acids depend on the length of the nucleic acids and the degree of complementarity, variables well known in the art. Typically, the hybridizable nucleic acid is 8 nucleotides or more in length (e.g., 10 nucleotides or more, 12 nucleotides or more, 15 nucleotides or more, 20 nucleotides or more, 22 nucleotides or more, 25 nucleotides or more, or 30 nucleotides or more).

It will be appreciated that the sequence of a polynucleotide need not be 100% complementary to the sequence of its target nucleic acid to specifically hybridize. Polynucleotides may comprise 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence complementarity to a target region in a target nucleic acid sequence to which it hybridizes.

Hybridization of the target sequence to the gRNA means that at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the target sequence and the nucleic acid sequence of the gRNA can hybridize to form a complex, or that at least 12, 15, 16, 17, 18, 19, 20, 21, 22 or more bases of the target sequence and the nucleic acid sequence of the gRNA can be complementarily paired and hybridized to form a complex.

Expression of

As used herein, the term "expression" refers to a process whereby a polynucleotide is transcribed from a DNA template (e.g., into mRNA or other RNA transcript) and/or a process whereby the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. Transcripts and encoded polypeptides may be collectively referred to as "gene products". If the polynucleotide is derived from genomic DNA, expression may include splicing of mRNA in eukaryotic cells.

Joint

As used herein, the term "linker" refers to a linear polypeptide formed from multiple amino acid residues joined by peptide bonds. The linker of the invention may be an amino acid sequence that is synthesized artificially, or a naturally occurring polypeptide sequence, such as a polypeptide having the function of a hinge region. Such linker polypeptides are well known in the art (see, e.g., holliger, P. Et al (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; poljak, R.J. Et al (1994) Structure 2:1121-1123).

Treatment of

As used herein, the term "treating" refers to treating or curing a disorder, delaying the onset of symptoms of a disorder, and/or delaying the progression of a disorder.

A subject

As used herein, the term "subject" includes, but is not limited to, various animals, plants, and microorganisms.

Animals

Such as mammals, e.g., bovine, equine, ovine, porcine, canine, feline, lagomorph (e.g., mice or rats), non-human primate (e.g., macaque or cynomolgus) or human. In certain embodiments, the subject (e.g., human) has a disorder (e.g., a disorder resulting from a disease-related gene defect).

Plants and methods of making the same

The term "plant" is understood to mean any differentiated multicellular organism capable of photosynthesis, including crop plants at any stage of maturity or development, in particular monocotyledonous or dicotyledonous plants, vegetable crops, including artichoke, broccoli, sesame seed, leek, asparagus, lettuce (e.g., head lettuce, leaf lettuce), chinese cabbage (bok choy), yellow arrowroot, melons (e.g., melon, watermelon, crohawa, white melon, cantaloupe), rape crops (e.g., cabbage, broccoli, chinese cabbage, kohlrabi, chinese cabbage), artichoke, carrot, cabbage (napa), okra, onion, celery, parsley, chick pea, parsnip, chicory, pepper, potato, cucurbit (e.g., zucchini, cucumber, zucchini, melon, pumpkin), radish, dried onion, turnip cabbage, purple eggplant (also known as eggplant), salon, chicory, green onion, chicory, garlic, spinach, green onion, melon, green vegetables (greens), beet (sugar beet and fodder beet), sweet potato, lettuce, horseradish, tomato, turnip, and spice; fruit and/or vine crops, such as apples, apricots, cherries, nectarines, peaches, pears, plums, prunes, cherries, quince, almonds, chestnuts, hazelnuts, pecans, pistachios, walnuts, oranges, blueberries, boysenberries (boysenberry), rubus parviflora, currants, rozerland, raspberries, strawberries, blackberries, grapes, avocados, bananas, kiwi fruits, persimmon, pomegranate, pineapple, tropical fruit, pear, melon, mango, papaya, and litchi, field crops such as clover, alfalfa, evening primrose, white mango, corn/maize (forage corn, sweet corn, popcorn), hops, jojoba, peanut, rice, safflower, small grain crops (barley, oat, rye, wheat, etc.), sorghum, tobacco, kapok, legumes (beans, lentils, peas, soybeans), oleaginous plants (rape, mustard, poppy, olives, sunflower, coconut, castor oil plants, cocoa beans, groundnut), arabidopsis, fiber plants (cotton, flax, jute), camphoraceae (cinnamon, camphor), or a plant such as coffee, sugarcane, tea, and natural rubber plants, and/or flower pot plants such as flowering plants, cactus, fleshy plants, and/or ornamental plants, and trees such as forests (leaf and evergreen trees, such as conifers), fruit trees, ornamental trees, and knotnuts (nu-tree), and other seedlings and tree-bearing and other plants.

Advantageous effects of the invention

The application discovers a Cas enzyme, and Blast results show that the Cas enzyme has lower consistency with the reported Cas enzyme, belongs to a Cas protein, and has wide application prospect.

Embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are only for illustrating the present invention and are not to be construed as limiting the scope of the present invention. Various objects and advantageous aspects of the present invention will become apparent to those skilled in the art from the following detailed description of the preferred embodiments and the accompanying drawings.

Sequence listing

Sequence number (SEQ ID No.)	Sequence description
		1	CasN-1 amino acid sequence
2	CasN-3 amino acid sequence
		3	CasN-4 amino acid sequence
4	CasN-6 amino acid sequence
		5	CasN-7 amino acid sequence
6	CasN-1 nucleotide sequence
		7	CasN-3 nucleotide sequence
8	CasN-4 nucleotide sequence
		9	CasN-6 nucleotide sequence
10	CasN-7 nucleotide sequence
		11	CasN-1 crRNA pairing region sequence
12	CasN-3 crRNA pairing region sequence
		13	CasN-4 crRNA pairing region sequence
14	CasN-6 crRNA pairing region sequence
		15	CasN-7 crRNA pairing region sequence
16	CrRNA pairing region sequence
		17	TracrRNA sequences

Drawings

FIG. 1-phylogenetic relationship of CasN with other proteins.

FIG. 2. Conserved domains of CasN.

FIG. 3. CrRNA pairing region sequence of CasN.

The trans-cleavage activity of CasN-1, casN-3, casN-4, casN-6, casN-7 is shown in FIG. 4.

Detailed Description

The following examples are only intended to illustrate the invention and are not intended to limit it. The experiments and methods described in the examples were performed substantially in accordance with conventional methods well known in the art and described in various references unless specifically indicated. For example, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cytobiology, genomics, and recombinant DNA used in the present invention can be found in Sammbruk (Sambrook), fries (Fritsch) and Meniere's (Maniatis), molecular cloning: laboratory Manual (MOLECULAR CLONING: A LABORATORY MANUAL), 2 nd time edition (1989), contemporary molecular biology laboratory Manual (CURRENT PROTOCOLS IN MOLECULAR BIOLOGY) (F.OMA.Ausubel et al edition (1987)), enzyme method (METHODS IN ENZYMOLOGY) series (academic Press): PCR 2:Utility method (PCR 2:A PRACTICAL APPROACH) (M.J. MaxPherson), B.D. Black. Hames) and G.R. Taylor. Edit (1995)), harrow (Harrow.53) and French (1987) in CULTURE (LTI.57) (Mannheil.1987).

In addition, the specific conditions are not specified in the examples, and the process is carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention. Those skilled in the art will appreciate that the examples describe the invention by way of example and are not intended to limit the scope of the invention as claimed. All publications and other references mentioned herein are incorporated by reference in their entirety.

EXAMPLE 1 Cas protein acquisition

The inventor analyzes a metagenome of an uncultured substance, and identifies a new class of Cas enzyme through redundancy removal and protein clustering analysis. Blast results show that Cas proteins have low sequence identity to the reported Cas proteins, and by building a evolutionary tree with Cas enzymes of other classes, these Cas enzymes were found to belong very significantly to an independent evolutionary branch and are further away from other proteins, as shown in fig. 1. The present invention designates this type of protein as CasN. The 5 CasN proteins obtained in this embodiment are designated CasN-1, 3, 4, 6, and 7, respectively, and the amino acid sequences and nucleic acid sequences of the proteins are shown in tables 2 to 3.

TABLE 2 amino acid sequences of different Cas proteins

TABLE 3 nucleic acid sequences of different Cas proteins

Structural analysis of CasN proteins shows that the CasN proteins comprise some conserved domains, specifically, the partial amino acid sequences of the RuvC domains of the CasN proteins are [ D/K/N/P ] N [ F/L/Y ] [ A/G/L/P/Q/V ] [ D/F/H/Y ] E [ I/L/T/V ] [ A/V ] [ H/K/N/R/T/V/W ] [ A/Q/R ] [ I/L/M ] [ I/L/V ] and [ A/M/Y ] [ F/I/L/M/S/V/Y ] [ I/V ] [ A/C/G ] [ I/L/V ] E [ D/H/N/R/T ] [ A/G/T ]. Wherein the above two conserved motifs CasN-1, casN-3, casN-4, casN-6, casN-7 are [ D/N/P ] N [ Y ] [ A/V ] [ D/F/Y ] E [ T/V ] [ A/V ] [ H/K/T ] [ A ] [ I/L/M ] [ I/V ] and [ A/M ] [ I/L/M/V ] [ I ] [ G ] [ I/L ] E [ D/H/R ] [ A/G/T ], as shown in FIG. 2 (the parts within the blocks are conserved motifs). The gene structure of CasN system is CasN-tracrRNA-crRNA, crRNA has strong conservation, as shown in figure 3, and the sequence of crRNA pairing region of CasN protein is shown in table 4.

TABLE 4 pairing region sequences of crRNA and tracrRNA corresponding to CasN

The CasN system may also employ other pairing region sequences such as:

GUCGGAUCACUGAGCGAGCGAUCUGAGAAGUGGCAC(SEQ ID No.16)。

EXAMPLE 2 verification of CasN protein Activity

This example was tested in vitro to verify the trans-cleavage activity of CasN-1, casN-3, casN-4, casN-6, casN-7. In this example, the gRNA capable of pairing with the target nucleic acid is used to guide CasN protein to recognize and bind to the target nucleic acid, then, casN protein excites trans-cleavage activity on any single-stranded nucleic acid, thereby cleaving the single-stranded nucleic acid detector in the system, fluorescent groups and quenching groups are respectively arranged at two ends of the single-stranded nucleic acid detector, fluorescence is excited if the single-stranded nucleic acid detector is cleaved, and in other embodiments, two ends of the single-stranded nucleic acid detector can be provided with labels capable of being detected by colloidal gold.

In this example, the target nucleic acid was selected to be single-stranded DNA, and N-B-i3g1-ssDNA0 was selected to have a sequence of CGACATTCCGAAGAACGCTGAAGCGCTGGGGGCAAATTGTGCAATTTGCGGC;

the tracrRNA sequence is:

GUCUAAAGGACAGAAUUUUUCAACGGGUGUGCCAAUGGCCACUUUCCAGGUG GCAAAGCCCGUUGAACUUCUCAAAAAGAACGCUCGCUCAGUGUUCUGAC(SEQ IDNo.17);

The crRNA sequence is:

GUCGGAUCACUGAGCGAGCGAUCUGAGAAGUGGCACCCCCCAGCGCUUCAGCG UUC (underlined region is the region of crRNA that targets the target nucleic acid);

The sequence of the single-stranded nucleic acid detector is FAM-TTATT-BHQ1;

a reaction system was used with a final Cas concentration of 50nM, a final tracrrna concentration of 100nM, a final crRNA concentration of 100nM, a final target nucleic acid concentration of 100nM, and a final single-stranded nucleic acid detector concentration of 200nM. Incubation at 37 ℃ and FAM fluorescence/30 s read. The control group had no target nucleic acid added.

As shown in FIG. 4, fluorescence was rapidly reported from single stranded nucleic acid detectors in CasN-1, casN-3, casN-4, casN-6, casN-7 cleavage systems in the presence of target nucleic acid, as compared to controls without target nucleic acid. The above experiments revealed that CasN-1, casN-3, casN-4, casN-6, casN-7, in combination with single-stranded nucleic acid detectors, can be used for detection of target nucleic acids. In FIG. 1,2, 3,4 and 5 are the experimental results of CasN-1, casN-3, casN-4, casN-6 and CasN-7, respectively, and 6, 7, 8, 9 and 10 are the control groups without target nucleic acid, casN-1, casN-3, casN-4, casN-6 and CasN-7, respectively.

While specific embodiments of the invention have been described in detail, it will be appreciated by those skilled in the art that various modifications and alternatives to those details could be developed in light of the overall teachings of the disclosure and that such modifications would be within the scope of the invention. The full scope of the invention is given by the appended claims together with any equivalents thereof.

Claims

1. A Cas protein, characterized in that the Cas protein is a CasN protein, and the CasN protein comprises the following two conserved domains:

The first conserved domain: X1NX2X3X4EX5X6X7X8X9X10; wherein, X1=D/K/N/P, X2=F/L/Y, X3=A/G/L/P/Q/V, X4=D/F/H/Y, X5=I/L/T/V, X6=A/V, X7=H/K/N/R/T/V/W, X8= A/Q/R, X9=I/L/M, X10=I/L/V;

The second conserved domain: X11X12X13X14X15EX16X17; wherein, X11=A/M/Y, X12=F/I/L/M/S/V/Y, X13=I/V, X14=A/C/G, X15=I/L/V, X16=D/H/N/R/Y, X17=A/G/T;

The amino acid sequence of the CasN protein is shown in SEQ ID No.1.

2. A fusion protein, comprising the Cas protein according to claim 1 and other modified parts, wherein the modified parts are selected from epitope tags, reporter gene sequences, and nuclear localization signal sequences.

3. An isolated polynucleotide, characterized in that the polynucleotide is a polynucleotide sequence encoding the Cas protein according to claim 1, or a polynucleotide sequence encoding the fusion protein according to claim 2.

4. A vector, characterized in that the vector comprises the polynucleotide according to claim 3 and a regulatory element operably linked thereto.

5. A CRISPR-Cas system, characterized in that the system comprises the Cas protein according to claim 1 and at least one gRNA capable of binding to the Cas protein, wherein the gRNA comprises a region binding to the Cas protein and a targeting sequence for a targeting nucleic acid.

6. A vector system, characterized in that the vector system comprises one or more vectors, and the one or more vectors comprise:

a) a first regulatory element, which is operably connected to a gRNA, wherein the gRNA comprises a region that binds to the Cas protein according to claim 1 and a targeting sequence that targets a nucleic acid;

b) a second regulatory element, which is operably linked to the Cas protein of claim 1;

Components (a) and (b) are located on the same or different carriers of the system.

7. A composition, characterized in that the composition comprises:

(i) a protein component selected from: the Cas protein according to claim 1 or the fusion protein according to claim 2;

(ii) a nucleic acid component selected from: a gRNA, or a nucleic acid encoding the gRNA, or a precursor RNA of the gRNA, or a precursor RNA nucleic acid encoding the gRNA; the gRNA comprises a region that binds to the Cas protein of claim 1 and a targeting sequence for a targeting nucleic acid;

The protein component and the nucleic acid component are combined with each other to form a complex.

8. An activated CRISPR complex, comprising:

(iii) binding to a target sequence on the gRNA.

9. An engineered host cell, which is a non-animal and plant cell, characterized in that 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 vector of claim 4, or the CRISPR-Cas system of claim 5, or the vector system of claim 6, or the composition of claim 7, or the activated CRISPR complex of claim 8.

10. Use of the Cas protein of claim 1, or the fusion protein of claim 2, or the polynucleotide of claim 3, or the vector of claim 4, or the CRISPR-Cas system of claim 5, or the vector system of claim 6, or the composition of claim 7, or the activated CRISPR complex of claim 8, or the host cell of claim 9 in gene targeting, wherein the application is for purposes other than disease diagnosis and treatment, and the subject of the application does not include humans; or, use in the preparation of a reagent or kit for gene targeting.

11. Use of the Cas protein of claim 1, or the fusion protein of claim 2, or the polynucleotide of claim 3, or the vector of claim 4, or the CRISPR-Cas system of claim 5, or the vector system of claim 6, or the composition of claim 7, or the activated CRISPR complex of claim 8, or the host cell of claim 9 in any one or more of the following, wherein the application is for non-disease diagnosis and treatment purposes, and the subject of the application does not include humans:

Non-specific cleavage and/or degradation of side branch nucleic acids; non-specific cleavage of single-stranded nucleic acids; nucleic acid detection.

12. A method for targeting a target nucleic acid, the method is for non-disease diagnosis and treatment purposes, the application object of the method does not include humans, 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 vector of claim 4, or the CRISPR-Cas system of claim 5, or the vector system of claim 6, or the composition of claim 7, or the activated CRISPR complex of claim 8, or the host cell of claim 9.

13. A method for cutting a single-stranded nucleic acid, the method is for non-disease diagnosis and treatment purposes, the application object of the method does not include humans, the method comprises contacting a nucleic acid population with the Cas protein and gRNA according to claim 1, the gRNA comprises a region that binds to the Cas protein according to claim 1 and a targeting sequence for targeting nucleic acid, wherein the nucleic acid population comprises a target nucleic acid and at least one non-target single-stranded nucleic acid, the gRNA is capable of targeting the target nucleic acid, and the Cas protein cuts the non-target single-stranded nucleic acid.

14. A kit for detecting a target nucleic acid in a sample, the kit comprising: (a) the Cas protein of claim 1, or a nucleic acid encoding the Cas protein; (b) gRNA, or a nucleic acid encoding the gRNA, or a precursor RNA comprising the gRNA, or a nucleic acid encoding the precursor RNA; the gRNA comprises a region that binds to the Cas protein of claim 1 and a targeting sequence for a targeting nucleic acid; and (c) a single-stranded nucleic acid detector that is single-stranded and does not hybridize with the gRNA.

15. A method for detecting a target nucleic acid in a sample, the method being a method for purposes other than disease diagnosis and treatment, the method being applicable to objects other than humans, the method comprising contacting the sample with the Cas protein, gRNA (guide RNA) and a single-stranded nucleic acid detector as described in claim 1, the gRNA comprising a region that binds to the Cas protein and a guide sequence that hybridizes with the target nucleic acid; detecting a detectable signal generated by the Cas protein cleaving the single-stranded nucleic acid detector, thereby detecting the target nucleic acid; the single-stranded nucleic acid detector does not hybridize with the gRNA.