CN114438055B - Novel CRISPR enzymes and systems and uses - Google Patents

Abstract

The invention belongs to the field of nucleic acid editing, and particularly relates to the technical field of regularly clustered spaced short palindromic repeats (CRISPR). Specifically, the invention provides a novel Cas enzyme, which is Cas-sf0005, has low homology with the reported Cas enzyme, can show nuclease activity in cells and outside the cells, and has wide application prospect.

Description

Novel CRISPR enzymes and systems and applications

technical field

The present invention relates to the field of gene editing, in particular to the technical field of regularly clustered interspaced short palindromic repeats (CRISPR). Specifically, the present invention relates to a novel CRISPR enzyme (or referred to as CRISPR protein, Cas effector protein, Cas enzyme or Cas protein), fusion proteins comprising such proteins, and nucleic acid molecules encoding them. The invention also relates to complexes and compositions for nucleic acid editing (eg, gene or genome editing) comprising the Cas proteins or fusion proteins of the invention, or nucleic acid molecules encoding them.

Background technique

CRISPR/Cas technology is a widely used gene editing technology. It specifically binds target sequences on the genome through RNA guidance and cleaves DNA to generate double-strand breaks, and uses biological non-homologous end joining or homologous recombination for site-specific gene editing.

The CRISPR/Cas9 system is the most commonly used type II CRISPR system, which recognizes the PAM motif of 3'-NGG and blunt-ends the target sequence. CRISPR/Cas Type V system is a newly discovered CRISPR system, which has a 5'-TTN motif and performs sticky end cleavage of target sequences, such as Cpf1, C2c1, CasX, CasY. However, the different CRISPR/Cas that currently exist have different advantages and disadvantages. For example, Cas9, C2c1 and CasX all require two RNAs for guide RNA, while Cpf1 requires only one guide RNA and can be used for multiple gene editing. CasX has a size of 980 amino acids, while the common Cas9, C2c1, CasY and Cpf1 are usually around 1300 amino acids in size. In addition, the PAM sequences of Cas9, Cpf1, CasX, and CasY are more complex and diverse, and C2c1 recognizes the strict 5'-TTN, so its target site is easier to predict than other systems, thereby reducing potential off-target effects.

In conclusion, given that currently available CRISPR/Cas systems are limited by some defects, the development of a more robust and novel CRISPR/Cas system with good performance in many aspects is of great significance for the development of biotechnology.

SUMMARY OF THE INVENTION

After a lot of experiments and repeated explorations, the inventors of the present application unexpectedly discovered a new type of endonuclease (Cas enzyme). Based on this discovery, the present inventors developed a new CRISPR/Cas system and a gene editing method based on this system.

Cas effector protein

In one aspect, the present invention provides a Cas protein, which is an effector protein in the CRISPR/Cas system. In the present invention, it is called Cas-sf0005 (the amino acid sequence is shown in SEQ ID No. 1) .

In one embodiment, the Cas protein amino acid sequence is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 94% compared to SEQ ID No. 1 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity and substantially retain the biological function of the sequence from which it is derived.

In one embodiment, the Cas protein amino acid sequence has one or more amino acid substitutions, deletions or additions compared to SEQ ID No. 1; and substantially retains the biological function of the sequence from which it is derived; The substitution, deletion or addition of one or more amino acids includes substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids .

It is clear to those skilled in the art that the structure of a protein can be altered without adversely affecting its activity and functionality, for example, one or more conservative amino acid substitutions can be introduced into the amino acid sequence of a protein without affecting the activity and/or activity of the protein molecule. or three-dimensional structure adversely affected. Examples and embodiments of conservative amino acid substitutions will be apparent to those skilled in the art. Specifically, the amino acid residue can be substituted with another amino acid residue belonging to the same group as the site to be substituted, that is, another non-polar amino acid residue can be substituted with a non-polar amino acid residue, and polar and uncharged An amino acid residue that is substituted for another polar, uncharged amino acid residue, a basic amino acid residue for another basic amino acid residue, and an acidic amino acid residue for another acidic amino acid residue. Such substituted amino acid residues may or may not be encoded by the genetic code. Conservative substitutions in which one amino acid is replaced by other amino acids belonging to the same group are within the scope of the present invention as long as the substitution does not result in inactivation of the biological activity of the protein. Therefore, the protein of the present invention may contain one or more conservative substitutions in the amino acid sequence, and these conservative substitutions are preferably generated by substitution according to Table 1. In addition, the present invention also encompasses proteins that also contain one or more other non-conservative substitutions, so long as 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 can be made at one or more predicted non-essential amino acid residues. "Non-essential" amino acid residues are amino acid residues that can be altered (deletion, substitution or substitution) without altering biological activity, while "essential" amino acid residues are required for biological activity. "Conservative amino acid substitutions" are substitutions in which amino acid residues are replaced with amino acid residues having similar side chains. Amino acid substitutions can be made in the non-conserved regions of the Cas protein described above. Generally, such substitutions are not made to conserved amino acid residues, or to amino acid residues located within conserved motifs, where such residues are required for protein activity. However, those skilled in the art will appreciate that functional variants may have minor conservative or non-conservative changes in conserved regions.

Table 1

initial residue 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 can be altered (substituted, deleted, truncated or inserted) from the N and/or C terminus of a protein and still retain its functional activity. Accordingly, proteins in which one or more amino acid residues are altered from the N and/or C terminus of a Cas protein while retaining its desired functional activity are also within the scope of the present invention. These changes may include those introduced by modern molecular methods such as PCR, including PCR amplifications that alter or extend protein-coding sequences by including amino acid-coding sequences in oligonucleotides used in PCR amplifications.

It will be appreciated that proteins can be altered in various ways, including amino acid substitutions, deletions, truncations, and insertions, and methods for such manipulations are generally known in the art. For example, amino acid sequence variants of the above proteins can be prepared by mutation of DNA. It can also be accomplished by other forms of mutagenesis and/or by directed evolution, e.g., single or multiple amino acid substitutions, using known mutagenesis, recombination and/or shuffling methods, in conjunction with relevant screening methods, Deletions and/or insertions.

Those skilled in the art will appreciate that these minor amino acid changes in the Cas proteins of the present invention can occur (eg, naturally occurring mutations) or produced (eg, 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 properties of the polypeptide can be altered, but the polypeptide can retain its activity. Smaller effects can be expected if the mutations present are not in close proximity to the catalytic domain, active site, or other functional domain.

Those skilled in the art can identify the essential amino acids of the Cas protein of the present invention according to methods known in the art, such as site-directed mutagenesis or protein evolution or analysis of bioinformatics. The catalytic domain, active site, or other functional domain 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 conjunction with putative key sites. point amino acid mutation.

In one embodiment, the Cas protein contains the amino acid sequence shown in SEQ ID No.1.

In one embodiment, the Cas protein is the amino acid sequence shown in SEQ ID No.1.

In one embodiment, the Cas protein is a derivatized protein with the same biological function as the protein having the sequence shown in SEQ ID No. 1.

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

The present invention also provides a fusion protein comprising the above-mentioned Cas protein and other modified parts.

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

In one embodiment, the modifying moiety is selected from the group consisting of epitope tags, reporter gene sequences, nuclear localization signal (NLS) sequences, targeting moieties, transcriptional activation domains (eg, VP64), transcriptional repression domains (eg, KRAB) domain or SID domain), a nuclease domain (eg, Fok1), and a domain having an activity selected from the group consisting of: nucleotide deaminase, methylase activity, demethylase, transcriptional activation activity, transcription inhibitory activity, transcription 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 other random combination. Such 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 at, near or near the terminus (eg, N-terminus, C-terminus or both) of the Cas protein of the invention.

The epitope tag (epitope tag) is well known to those skilled in the art, including but not limited to His, V5, FLAG, HA, Myc, VSV-G, Trx, etc., and those skilled in the art can select other suitable epitopes Labeling (eg, purification, detection, or tracking).

The reporter gene 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, and the like.

In one embodiment, the fusion protein of the present invention comprises a domain capable of binding to DNA molecules or intracellular molecules, such as maltose binding protein (MBP), DNA binding domain (DBD) of Lex A, DBD of GAL4, and the like.

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

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

In one embodiment, the modified moiety is directly attached to the N-terminus or C-terminus of the Cas protein of the invention.

In one embodiment, the modified moiety is linked to the N-terminus or C-terminus of the Cas protein of the invention via a linker. Such linkers are well known in the art, examples of which include, but are not limited to, include one or more (eg, 1, 2, 3, 4, or 5) amino acids (eg, Glu or Ser) or amino acid derivatives (eg, 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 its production method, for example, it can be produced by genetic engineering methods (recombinant technology) or chemical synthesis methods.

Nucleic acid of Cas protein

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

(a) a polynucleotide sequence encoding the Cas protein or fusion protein of the present invention;

(b) a polynucleotide whose sequence is shown in SEQ ID No. 2 or 3;

(c) having one or more base substitutions, deletions or additions (e.g. 1, 2, 3, 4, 5, 6, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 base substitutions, deletions or additions) sequences;

(d) The homology between the nucleotide sequence and the sequence shown in SEQ ID No. 2 or 3 is ≥80% (preferably ≥90%, more preferably ≥95%, optimally ≥98%), and encodes The polynucleotide of the polypeptide shown in SEQ ID No. 1; or,

(e) a polynucleotide complementary to any of the polynucleotides of (a)-(d).

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

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

Direct Repeat sequence

In another aspect, the present invention provides an engineered direct repeat sequence that forms a complex with the above Cas protein.

The direct repeat sequence is linked with a guide sequence capable of hybridizing with the target sequence to form a guide RNA (guideRNA or gRNA).

The hybridization of the target sequence and the gRNA represents at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% of the target sequence and the guide sequence of the gRNA , 97%, 98%, 99%, or 100% identity, so that it can hybridize to form a complex; or at least 12, 15, 16, 17, 18 guide sequences representing the target sequence and the gRNA, 19, 20, 21, 22, or more bases can complementarily pair to form a complex.

In some embodiments, the direct repeat sequence is at least 90% identical to the sequence set forth in SEQ ID No. 4 or 5. In some embodiments, the direct repeat sequence has one or more base substitutions, deletions or additions (eg 1, 2, 3, 4) compared to the sequence shown in SEQ ID No. 4 or 5 1, 5, 6, 7, 8, 9 or 10 base substitutions, deletions or additions).

In some embodiments, the direct repeat is shown in SEQ ID No. 4 or 5.

guide RNA (gRNA)

In another aspect, the present invention provides a gRNA, the gRNA includes a first segment and a second segment; the first segment is also referred to as "skeleton region", "protein binding segment", "protein binding segment" Sequence", or "DirectRepeat sequence"; the second segment is also referred to as "targeting sequence targeting nucleic acid" or "targeting segment targeting nucleic acid", or "targeting target sequence" boot sequence".

The first segment of the gRNA is capable of interacting with the Cas protein of the present invention, thereby allowing the Cas protein and the gRNA to form a complex.

In a preferred embodiment, the first segment is a direct repeat as described above.

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

The percent complementarity between the targeting sequence of the targeting nucleic acid or the targeting segment of the targeting nucleic acid and the target sequence of the target nucleic acid can be at least 60% (eg, 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 "backbone region", "protein binding segment", "protein binding sequence", or "direct repeat sequence" of the gRNA of the present invention can interact with the CRISPR protein (or, the Cas protein). The gRNA of the present invention guides its interacting Cas protein to a specific nucleotide sequence in the target nucleic acid through the action of the targeting sequence of the targeting nucleic acid.

Preferably, the guide RNA comprises a first segment and a second segment from the 5' to 3' direction.

In the present invention, the second segment can also be understood as a guide sequence that hybridizes with the target sequence.

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

The gRNA of the Cas-sf0005 protein of the present invention comprises a guide sequence that hybridizes to a target nucleic acid, wherein the target nucleic acid comprises a sequence located at the 3' end of the protospacer adjacent motif (PAM); the aforementioned PAM sequence is 5'TBN- 3', where B=T/C/G and N=A/T/C/G.

carrier

The present invention also provides a vector comprising the above Cas protein, isolated nucleic acid molecule or polynucleotide; preferably, it further comprises a regulatory element operably linked therewith.

In one embodiment, the regulatory elements are selected from one or more of the group consisting of enhancers, transposons, promoters, terminators, leader sequences, polyadenylation sequences, marker genes.

In one embodiment, the vectors include cloning vectors, expression vectors, shuttle vectors, and integration vectors.

In some embodiments, the vectors included in the system are viral vectors (eg, retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated vectors, and herpes simplex vectors), but also plasmids, viruses, cosmids, Types of bacteriophage and the like, 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.

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

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

The one or more guide RNAs target one or more target sequences in the cell. The one or more target sequences hybridize to the genomic locus of the DNA molecule encoding one or more gene products, and direct the Cas protein to the genomic locus of the DNA molecule of the one or more gene products, the Cas protein The target sequence is modified, edited or cleaved after reaching the target sequence position, whereby the expression of the one or more gene products is altered or modified.

Cells of the present 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 present invention also provides an engineered non-naturally occurring vector system that may include one or more vectors comprising:

a) a first regulatory element operably linked to the gRNA,

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 (eg, constitutive or inducible promoters), enhancers (eg, 35S promoter or 35S enhanced promoter), internal ribosome entry sites (IRES), and others Expression control elements (eg, transcription termination signals such as polyadenylation signals and poly U sequences).

In some embodiments, the vectors in the system are viral vectors (eg, retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated vectors and herpes simplex vectors), but also plasmids, viruses, cosmids, phages and other types, 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, delivery systems are nanoparticles, liposomes, exosomes, microvesicles, and gene guns.

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 within the cell. In one embodiment, the target sequence is present in the nucleus or in the cytoplasm (eg, organelle). In one embodiment, the cell is a eukaryotic cell. In other embodiments, the cells are prokaryotic cells.

In one embodiment, the Cas protein is linked to one or more NLS sequences. 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 the 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 present invention relates to an engineered CRISPR system comprising the above Cas protein and one or more guide RNAs, wherein the guide RNAs comprise direct repeats and spacers capable of hybridizing to a target nucleic acid sequence, the Cas protein is capable of binding the guide RNA and targeting a target nucleic acid sequence complementary to a spacer sequence.

In one embodiment, when the target nucleic acid is DNA (preferably, double-stranded DNA), the target nucleic acid is located at the 3' end of a protospacer adjacent motif (PAM), and the PAM has a 5'TBN- The sequence shown at 3', where B=T/C/G and N=A/T/C/G.

Protein-nucleic acid complexes/compositions

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

(i) a protein component selected from the group consisting of the above-mentioned Cas proteins, derivatized proteins or fusion proteins, and any combination thereof; and

(ii) a nucleic acid component comprising (a) a leader sequence capable of hybridizing to a target sequence; and (b) a direct repeat sequence capable of binding to the Cas protein of the invention.

The protein component and the nucleic acid component combine with 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 complex

In another aspect, the present invention also provides an activated CRISPR complex, the activated CRISPR complex comprising: (1) a protein component selected from the group consisting of: a Cas protein, a derivatized protein or a fusion protein of the present invention, and any combination thereof; (2) a gRNA comprising (a) a guide sequence capable of hybridizing to a target sequence; and (b) a direct repeat sequence capable of binding to the Cas protein of the present invention; and (3) binding on the gRNA target sequence. Preferably, the binding is through the targeting sequence of the targeting nucleic acid on the gRNA and the target nucleic acid.

The term "activated CRISPR complex", "activation complex" or "ternary complex" as used herein refers to the complex in which Cas protein, gRNA and target nucleic acid are bound or modified in the CRISPR system.

The Cas protein and gRNA of the present invention can form a binary complex, and the binary complex is activated when combined with a nucleic acid substrate to form an activated CRISPR complex. The nucleic acid substrate and the spacer sequence in the gRNA (or referred to as , complementary to the leader sequence that hybridizes to the target nucleic acid. In some embodiments, the spacer sequence of the gRNA perfectly matches the target substrate. In other embodiments, the spacer sequence of the gRNA matches a portion (contiguous or non-contiguous) of the target substrate.

Delivery and Delivery Compositions

The Cas proteins, gRNAs, fusion proteins, nucleic acid molecules, vectors, systems, complexes and compositions of the present invention can be delivered by any method known in the art. Such methods include, but are not limited to, electroporation, lipofection, nucleofection, microinjection, sonoporation, gene gun, calcium phosphate mediated transfection, cationic transfection, lipofection, dendritic Transfection, heat shock transfection, nucleofection, magnetic transfection, lipofection, punch transfection, optical transfection, reagent-enhanced nucleic acid uptake, and via liposomes, immunoliposomes, viral particles, artificial viruses body delivery.

Therefore, in another aspect, the present invention provides a delivery composition comprising a delivery vehicle, and one or any of several selected from the group consisting of the Cas protein, fusion protein, nucleic acid molecule, vector, system, Compounds and compositions.

In one embodiment, the delivery vehicle is a particle.

In one embodiment, the delivery vehicle is selected from lipid particles, sugar particles, metal particles, protein particles, liposomes, exosomes, microvesicles, gene guns, or viral vectors (eg, replication-defective retroviruses , lentivirus, adenovirus or adeno-associated virus).

host cell

The present invention also relates to an in vitro, ex vivo or in vivo cell or cell line or their progeny, said cell or cell line or their progeny comprising: the Cas protein, fusion protein, nucleic acid of the present invention Molecules, protein-nucleic acid complexes, activated CRISPR complexes, vectors, delivery compositions of the invention.

In certain embodiments, the cells are prokaryotic cells.

In certain embodiments, the cells are eukaryotic cells. In certain embodiments, the cells are mammalian cells. In certain embodiments, the cells are human cells. In certain embodiments, the cells are non-human mammalian cells, eg, non-human primate, bovine, ovine, porcine, canine, monkey, rabbit, rodent (eg, rat or mouse) cells. In certain embodiments, the cells are non-mammalian eukaryotic cells, eg, cells of poultry birds (eg, chickens), fish, or crustaceans (eg, clams, shrimp). In certain embodiments, the cells are plant cells, such as cells possessed by monocotyledonous or dicotyledonous plants or cells possessed by cultivated plants or food crops such as cassava, corn, sorghum, soybean, wheat, oat, or rice, for example Algae, tree or producing plants, fruits or vegetables (eg, trees such as citrus trees, nut trees; nightshade, cotton, tobacco, tomatoes, grapes, coffee, cocoa, etc.).

In certain embodiments, the cells are stem cells or stem cell lines.

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

Gene editing methods and applications

The Cas protein, nucleic acid, the above-mentioned composition, the above-mentioned CIRSPR/Cas system, the above-mentioned vector system, the above-mentioned delivery composition or the above-mentioned activated CRISPR complex or the above-mentioned host cell of the present invention can be used for any or any of the following purposes: targeting and/or editing target nucleic acid; cutting double-stranded DNA, single-stranded DNA or single-stranded RNA; specifically editing double-stranded nucleic acid; base-editing double-stranded nucleic acid; base-editing single-stranded nucleic acid. In other embodiments, it can also be used to prepare reagents or kits for any or any of the above uses.

The present invention also provides the application of the above-mentioned Cas protein, nucleic acid, above-mentioned composition, above-mentioned CIRSPR/Cas system, above-mentioned vector system, above-mentioned delivery composition or above-mentioned activated CRISPR complex in gene editing, gene targeting or gene cutting; Alternatively, use in the preparation of reagents or kits for gene editing, gene targeting or gene cleavage.

In one embodiment, the gene editing, gene targeting or gene cleavage is intracellular and/or extracellular gene editing, gene targeting or gene cleavage.

The present invention also provides a method for editing a target nucleic acid, targeting a target nucleic acid or cutting a target nucleic acid, the method comprising combining the target nucleic acid with the above Cas protein, nucleic acid, the above composition, the above CIRSPR/Cas system, the above vector system, The above-mentioned delivery composition or the above-mentioned activated CRISPR complex is contacted. In one embodiment, the method is editing a target nucleic acid, targeting a target nucleic acid, or cleaving a target nucleic acid, either intracellularly or extracellularly.

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

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

On the other hand, the present invention also provides a kit for gene editing, gene targeting or gene cleavage, the kit includes the above Cas protein, gRNA, nucleic acid, the above composition, the above CIRSPR/Cas system, the above The vector system, the delivery composition described above, the activated CRISPR complex described above, or the host cell described above.

In another aspect, the invention provides the above-mentioned Cas protein, nucleic acid, above-mentioned composition, above-mentioned CIRSPR/Cas system, above-mentioned carrier system, above-mentioned delivery composition, above-mentioned activated CRISPR complex or above-mentioned host cell in the preparation of preparation or kit. Use, the formulation or kit is for:

(i) gene or genome editing;

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

(iii) editing target sequences in target loci to modify organisms or non-human organisms;

(iv) treatment of disease;

(iv) Targeting target genes.

Preferably, the above-mentioned gene or genome editing is intracellular or extracellular gene or genome editing.

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

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

Methods of specifically modifying target nucleic acids

In another aspect, the present invention also provides a method for specifically modifying a target nucleic acid, the method comprising: combining the target nucleic acid with the aforementioned Cas protein, nucleic acid, the aforementioned composition, the aforementioned CIRSPR/Cas system, the aforementioned carrier system, and the aforementioned delivery composition or contact with the activated CRISPR complex described above.

This specific modification can occur in vivo or in vitro.

This specific modification can occur intracellularly or extracellularly.

In some cases, the cells are selected from prokaryotic cells or eukaryotic cells, eg, animal cells, plant cells, or microbial cells.

In one embodiment, the modification refers to a break in the target sequence, eg, a single-strand/double-strand break in DNA, or a single-strand break in 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 the donor polynucleotide A portion of the copy is integrated into the target nucleic acid.

In one embodiment, the modification further comprises inserting an editing template (eg, exogenous nucleic acid) into the fragment.

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

Definition of Terms

In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the operation steps such as molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics and recombinant DNA used in this paper are conventional steps widely used in the corresponding fields. . Meanwhile, for a better understanding of the present invention, definitions and explanations of related terms are provided below.

Cas protein

In the present invention, Cas protein, Cas enzyme, and Cas effector protein can be used interchangeably; the inventors discovered and identified a Cas effector protein for the first time, which has an amino acid sequence selected from the following:

(i) SEQ ID No. 1;

(ii) having one or more amino acid substitutions, deletions or additions compared to the sequence shown in SEQ ID No. 1 (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions) sequences; or

(iii) at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 80%, at least 85%, at least 80%, at least 85%, Sequences of 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.

Nucleic acid cleavage or cleavage of nucleic acids herein includes: DNA or RNA cleavage (Cis cleavage) in a target nucleic acid produced by the Cas enzymes described herein, DNA or RNA cleavage in a side branch nucleic acid substrate (single-stranded nucleic acid substrate) ( 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 break or a single-stranded RNA break.

CRISPR system

As used herein, the terms "Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-Associated (Cas) (CRISPR-Cas) system" or "CRISPR system" are used interchangeably and are of skill in the art In the meaning commonly understood by the person, it generally includes a transcript or other element associated with the expression of a CRISPR-associated ("Cas") gene, or a transcript or other element capable of directing the activity of said Cas gene.

CRISPR/Cas complex

As used herein, the term "CRISPR/Cas complex" refers to a complex formed by the binding of a guide RNA (guide RNA) or mature crRNA to a Cas protein, comprising a guide sequence hybridized to a target sequence and bound to a Cas protein A protein-binding direct repeat that recognizes and cleaves polynucleotides that hybridize to the guide RNA or mature crRNA.

Guide RNA (guide RNA, gRNA)

As used herein, the terms "guide RNA (gRNA)", "mature crRNA", "guide sequence" are used interchangeably and have the meanings commonly understood by those skilled in the art. In general, a guide RNA may comprise a direct repeat and a guide sequence, or consist essentially of or consist of a direct repeat and a guide sequence.

In certain instances, a guide sequence is any polynucleotide sequence that is sufficiently complementary to a target sequence to hybridize to the target sequence and direct specific binding of the CRISPR/Cas complex to the target sequence. In one embodiment, when optimally aligned, the degree of complementarity between the guide 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%. Determining the optimal alignment is within the ability of one of ordinary skill in the art. For example, there are published and commercially available alignment algorithms and programs such as, but not limited to, ClustalW, Smith-Waterman in matlab, Bowtie, Geneious, Biopython, and SeqMan.

target sequence

"Target sequence" refers to a polynucleotide targeted by a guide sequence in a gRNA, eg, a sequence complementary to the guide sequence, wherein hybridization between the target sequence and the guide sequence will facilitate the CRISPR/Cas complex (including Cas protein and gRNA). Complete complementarity is not required, as long as there is sufficient complementarity to cause hybridization and facilitate the formation of a CRISPR/Cas complex.

The target sequence can comprise any polynucleotide, such as DNA or RNA. In certain instances, the target sequence is intracellular or extracellular. In certain instances, the target sequence is located in the nucleus or cytoplasm of the cell. In certain instances, the target sequence may be located in an organelle of the eukaryotic cell such as the mitochondria or chloroplast. A sequence or template that can be used for recombination into a target locus containing the target sequence is referred to as an "edited template" or "edited polynucleotide" or "edited sequence." 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 endogenous or exogenous to a cell (eg, a eukaryotic cell). For example, the target polynucleotide can be a polynucleotide present in the nucleus of a eukaryotic cell. The target polynucleotide can be a sequence encoding a gene product (eg, a protein) or a non-coding sequence (eg, a regulatory polynucleotide or unwanted DNA). In some cases, the target sequence should be associated with a protospacer adjacent motif (PAM).

Wild type

As used herein, the term "wild-type" has the meaning commonly understood by those skilled in the art, which denotes the typical form of an organism, strain, gene, or a characteristic that distinguishes it from a mutant or variant form as it occurs in nature, It can be isolated from sources in nature and has not been intentionally modified by humans.

derivatization

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

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

derivatized protein

Also known as "protein derivatives", refers to modified forms of proteins, eg, 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 the involvement of man. 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 with which they are associated in nature or as found in nature.

Orthologue (orthologue, ortholog)

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

identity

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 an adenine, or both A position in each of the polypeptides is occupied by a 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 compared x 100. For example, two sequences are 60% identical if 6 out of 10 positions match. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (matching at 3 positions out of a total of 6). Typically, comparisons are made when two sequences are aligned for maximum identity. Such alignment can be accomplished using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). The algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), can also be used, using the PAM120 weight residue table, A gap length penalty of 12 and a gap penalty of 4 were used to determine percent identity between two amino acid sequences. In addition, the algorithm of Needleman and Wunsch (J MoI Biol. 48:444-453 (1970)) in the GAP program integrated into the GCG software package (available at www.gcg.com), using the Blossum 62 matrix or PAM250 matrix with gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6 to determine percent identity between two amino acid sequences .

carrier

The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it is linked. 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 (eg, circular); nucleic acid molecules that include DNA, RNA, or both nucleic acid molecules; and a wide variety of other polynucleotides known in the art. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements carried by it can be expressed in the host cell. A vector can be introduced into a host cell to thereby produce transcripts, proteins, or peptides, including proteins, fusion proteins, isolated nucleic acid molecules, etc., as described herein (eg, CRISPR transcripts, such as nucleic acid transcripts). , protein, or enzyme). 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 an origin of replication site.

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

Another type of vector is a viral vector, in which virus-derived DNA or RNA sequences are present for packaging viruses (eg, retroviruses, replication-defective retroviruses, adenoviruses, replication-defective adenoviruses, and adeno-associated virus) vector. Viral vectors also contain polynucleotides carried by the virus used for transfection into a host cell. Certain vectors (eg, bacterial vectors with bacterial origins of replication and episomal mammalian vectors) are capable of autonomous replication in the host cell into which they are introduced.

Other vectors (eg, non-episomal mammalian vectors) integrate into the host cell's genome upon introduction into the host cell, and thus replicate with the host genome. Furthermore, 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 cell

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. coli or Bacillus subtilis, such as microbial cells, fungal cells, animal cells, and plants eukaryotic 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 (eg, transcription termination signals such as polyadenylation signals and poly U sequence), which is described in detail in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, CA State (1990). In certain instances, 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 only in certain host cells (eg, tissue-specific regulatory sequences). Tissue-specific promoters can primarily direct expression in the desired tissue of interest, such as muscle, neuron, bone, skin, blood, specific organs (eg, liver, pancreas), or specific cell types (eg, lymphocytes). In certain instances, regulatory elements may also direct expression in a timing-dependent manner (eg, 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-U 5' fragment in the LTR of HTLV-1 ((Mol. Cell. Biol., p. 8( 1) Vol, pp. 466-472, 1988); the SV40 enhancer; and the intron sequence between exons 2 and 3 of rabbit β-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), pp. 1527-31, 1981).

Promoter

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

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, ie, a protein with an NLS is transported to the nucleus. Typically, NLSs contain positively charged Lys or Arg residues exposed on the protein surface. Exemplary nuclear localization sequences include, but are not limited to, NLS from the following: SV40 large T antigen, EGL-13, c-Myc, and TUS proteins. In some embodiments, the NLS comprises the 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 sequence MSRRRKANPTKLSENAKKLAKEVEN. 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, the sequence KIPIK in the yeast transcriptional repressor Matα2, and PY-NLS.

operatively connected

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 (e.g., , in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).

complementarity

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 conventional Watson-Crick or other non-traditional types. The percent complementarity represents the percentage of residues in a nucleic acid molecule that can hydrogen bond (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8 out of 10). , 9, 10 are 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Perfectly complementary" means that all contiguous residues of one nucleic acid sequence hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. "Substantially complementary" as used herein means in a At least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98 over a region of 30, 35, 40, 45, 50 or more nucleotides %, 99%, or 100% degree of complementarity, alternatively, refers to two nucleic acids that hybridize under stringent conditions.

Stringent conditions

As used herein, "stringent conditions" for hybridization refer 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 generally sequence-dependent and 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.

hybrid

The terms "hybrid" or "complementary" or "substantially complementary" mean that a nucleic acid (eg, RNA, DNA) comprises a nucleotide sequence that enables it to bind non-covalently, i.e. in a sequence-specific, anti-parallel fashion ( That is, a nucleic acid that specifically binds a complementary nucleic acid) forms base-pairing and/or G/U base-pairing, "anneals" or "hybridizes" with another nucleic acid.

Hybridization requires that the two nucleic acids contain complementary sequences, although there may be mismatches between the bases. Suitable conditions for hybridization between two nucleic acids depend on the length and degree of complementarity of the nucleic acids, variables well known in the art. Typically, hybridizable nucleic acids are 8 nucleotides or more in length (eg, 10 nucleotides or more, 12 nucleotides or more, 15 nucleotides or more, 20 nucleotides or more) 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 hybridize specifically. The polynucleotide may comprise 60% or higher, 65% or higher, 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher higher, 98% or higher, 99% or higher, 99.5% or higher, or the sequence complementarity of the target region in the target nucleic acid sequence to which it hybridizes is 100%.

Hybridization of the target sequence to the gRNA represents at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% of the nucleic acid sequence of the target sequence and the gRNA , 97%, 98%, 99% or 100% can hybridize to form a complex; or at least 12, 15, 16, 17, 18, 19, 20 nucleic acid sequences representing the target sequence and the gRNA 1, 21, 22 or more bases can complementarily pair and hybridize to form a complex.

Express

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

joint

As used herein, the term "linker" refers to a linear polypeptide formed by connecting multiple amino acid residues by peptide bonds. The linker of the present invention may be a synthetic amino acid sequence, or a naturally occurring polypeptide sequence, such as a polypeptide having hinge region function. 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 ).

treat

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

subject

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

animal

For example mammals such as bovines, equines, ovines, porcines, canines, felines, lagomorphs, rodents (eg, mice or rats), non-human primates Animals (eg, macaques or cynomolgus monkeys) or humans. In certain embodiments, the subject (eg, human) has a disorder (eg, a disorder caused by a disease-related gene defect).

plant

The term "plant" is to be understood as 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 artichokes, cabbage, Arugula, leeks, asparagus, lettuce (eg, head lettuce, leaf lettuce, romaine), bok choy, yellow taro, melons (eg, melon, watermelon, crenshaw ), white melon, romaine melon), canola crops (e.g., Brussels sprouts, cabbage, cauliflower, broccoli, kale, kale, Chinese cabbage, pak choi), spinach, carrots, napa, Okra, onions, celery, parsley, chickpeas, parsnips, chicory, peppers, potatoes, gourds (e.g., zucchini, cucumbers, zucchini, zucchini, squash), radishes, dried bulb onions, rutabagas, Purple eggplant (also called eggplant), salsify, endive, shallots, endive, garlic, spinach, green onions, zucchini, greens, beets (sugar beets and fodder beets), sweet potatoes, chard, Wasabi, tomatoes, turnips, and spices; fruits and/or trailing crops such as apples, apricots, cherries, nectarines, peaches, pears, plums, prunes, cherries, quinces, almonds, chestnuts, hazelnuts, pecans, Pistachios, walnuts, citrus, blueberries, boysenberry, cranberries, currant, loganberry, raspberry, strawberry, blackberry, grape, avocado, banana, kiwi, persimmon, pomegranate, pineapple , tropical fruits, pomes, melons, mangoes, papayas, and lychees; field crops such as clover, alfalfa, evening primrose, jasmine, corn/maize (fodder corn, sweet corn, popcorn), hops, lotus Barley, peanuts, rice, safflower, small grain crops (barley, oats, rye, wheat, etc.), sorghum, tobacco, kapok, legumes (beans, lentils, peas, soybeans), oily plants (rapese, Mustard greens, olives, sunflowers, coconuts, castor oil plants, cocoa beans, groundnuts), Arabidopsis, fibrous plants (cotton, flax, jute), lauraceae (cinnamon, camphor), or a plant such as coffee, sugarcane , tea, and natural rubber plants; and/or flower bed plants, such as flowering plants, cacti, succulents, and/or ornamentals, and trees such as forests (broadleaf and evergreen, such as conifers), fruit trees, ornamental trees, and fruit trees Nut-bearing trees, as well as shrubs and other seedlings.

Beneficial Effects of Invention

A new type of Cas enzyme has been discovered in the present invention. Blast results show that the Cas enzyme of the present application has low consistency with the reported Cas enzyme, and it can exhibit nuclease activity in vivo and in vitro, and has broad application prospects. .

The embodiments of the present invention will be described in detail below with reference to the drawings and examples, but those skilled in the art will understand that the following drawings and examples are only used to illustrate the present invention, rather than limit 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 accompanying drawings and the following detailed description of the preferred embodiments.

Description of drawings

Figure 1. PAM structure of Cas-sf0005.

Figure 2. The cleavage of double-stranded nucleic acid by Cas-sf0005.

Figure 3. Sequencing of target genes edited by Cas-sf0005 in eukaryotic cells.

sequence information

SEQ ID No. describe 1 Amino acid sequence of Cas-sf0005 2 Nucleic acid sequence encoding Cas-sf0005 3 Nucleic acid sequence of humanized Cas-sf0005 4 DR region of gRNA of Cas-sf0005 5 DR region of gRNA of Cas-sf0005

Detailed ways

The following examples are only used to describe the present invention, but not to limit the present invention. Unless otherwise indicated, the experiments and methods described in the Examples were performed essentially according to conventional methods well known in the art and described in various references. For example, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA used in the present invention can be found in Sambrook, Fritsch ) and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd ed. (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY ) (F.M. Ausubel et al., (1987)); "METHODS IN ENZYMOLOGY" series (Academic Publishing Company): "PCR 2: A PRACTICAL APPROACH" ) (edited by M.J. MacPherson, B.D. Hames and G.R. Taylor (1995), edited by Harlow and Lane (1988) Antibodies: ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (ed. R.I. Freshney (1987)).

In addition, if the specific conditions are not indicated in the examples, it is carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used without the manufacturer's indication are conventional products that can be obtained from the market. Those skilled in the art 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. Acquisition of Cas protein

The inventors analyzed the uncultured metagenome, and identified a new Cas enzyme through de-redundancy and protein cluster analysis. The Blast results showed that the Cas protein had a low sequence identity with the reported Cas protein, and was named Cas-sf0005 in the present invention.

The amino acid sequence of the above-mentioned protein, the nucleic acid sequence encoding the nucleic acid sequence and the optimized nucleic acid sequence of human codons are as follows:

SEQ ID No. describe 1 Amino acid sequence of Cas-sf0005 2 Nucleic acid sequence encoding Cas-sf0005 3 Nucleic acid sequence of humanized Cas-sf0005

The direct repeat sequence of the gRNA corresponding to the above protein was analyzed, and the results showed:

The direct repeat sequence of the gRNA corresponding to the Cas-sf0005 protein is CCGUCAACGUUCAACGCUUGCUCGGUUCGCCGAGAC (SEQ ID No. 4) or CCGUCAACGUUCAACGCUUGCUCGGUACGCCGAGAC (SEQ ID No. 5).

Example 2. Identification of the PAM domain of Cas-sf0005 protein

A PAM library was constructed, and the synthetic sequence was: CGTGTTTCGTAAAGTCTGGAAACGCGGAAGCCCCCAGCGCTTTCAGCGTTCNNNNNN TCCCCTACGTGCTGCTGAAGTTGC CCGCAA, N is a random deoxynucleotide, and the target sequence is underlined. The pacyc184 vector was ligated into the pacyc184 vector after filling with Klenow enzyme. After transformation of E. coli, the plasmid was extracted to form a PAM library. The sequence of gRNA Cas-sf0005-5'spacer1 20bp is: CCGUCAACGUUCAACGCUUGCUCGGUUCGCCGAGAC UCCCCUACGUGC UGCUGAAG (the underlined area is the targeting area);

Primer sequence: TK-117: CGGCATTCCTGCTGAACCGCTCTTCCGATCT;

TK-111:GATCGGAAGAGCGGTTCAGCAGGAATGCCG;

PAM-after-F: ACTCAGGGGTCTTCGGTTTCCGTGTT;

S6-PAM-after: ACTCAGCTGAACCGCTCTTCCG;

Obtain Cas-sf0005 protein preference PAM library: 50nM Cas-sf0005 protein, 50nM gRNA in buffer 25℃, incubate for 10min. Add PAM library plasmid (10ng/μL), incubate at 37°C for 1h; incubate at 85°C for 20min. Add 2.5U DreamTaq DNA Polymerase (5 U/µL) (Thermo Fisher Scientific) and 4µL 2.5mM dNTP Mix (full gold) to the above system, incubate at 72°C for 30min, fill in the cleaved end and add A at the 3' end. The above products were purified by a kit (Omega Gel Extraction Kit D2500). The primers TK-117 and TK-111 were annealed and ligated with the above product by T4 ligase. The obtained ligation product was subjected to PCR reaction with primers PAM-after-F and S6-PAM-after to obtain the Cas-sf0005 protein preference PAM library. The PCR product was subjected to second-generation sequencing, and the PAM sequence was obtained after analysis. As shown in 1, the PAM sequence recognized by Cas-sf0005 is 5'TBN-3', where B=T/C/G, N=A/T/C/G.

Example 3. Application of Cas-sf0005 protein in double-stranded nucleic acid editing

In this example, the cleavage activity of Cas-sf0005 protein on double-stranded DNA was tested by in vitro detection. In this example, a gRNA that can pair with the target nucleic acid is used to guide the Cas-protein to recognize and bind to the double-stranded target nucleic acid; then, the Cas protein stimulates the cleavage activity of the double-stranded target nucleic acid, thereby cutting the double-stranded target nucleic acid in the system. The cleaved double-stranded target nucleic acid is detected by agarose double-stranded electrophoresis.

In this example, the target nucleic acid is selected to be double-stranded DNA, and its sequence is: GAACGCTGAAGCGCTGGGGGCA TTA TCCC CTACGTGCTGCTGAAG TTGC is connected to the vector T-Vector-pEASY-Blunt Simple Cloning Vector; the italicized part of TTA is the PAM sequence, and the underlined area is the targeting region. gRNA-Cas-sf0005-5'spacer1-20bp: CCGUCAACGUUCAACGCUUGCUCGGUUCGCCGAGAC UCCCCUACGUGCUGCUGAAG (the underlined area is the targeting area); the following reaction system is used: the final concentration of Cas-sf0005 is 50nM, the final concentration of gRNA is 500nM, and the final concentration of double-stranded target nucleic acid is 5ng /µL. Cas protein and gRNA were incubated at 25°C for 10 min; double-stranded target nucleic acid was added, and incubated at 37°C for 1 h. 1.0% agarose electrophoresis detection. The experimental group was supplemented with target nucleic acid and gRNA, while the control group was not supplemented with gRNA.

As shown in Figure 2, lane 1 is the experimental group, lane 2 is the control group, and lane 3 is the Trans2K plus DNA marker. Compared with the control without gRNA, Cas-sf0005 can effectively cut the double-stranded nucleic acid in the system in the experimental group containing target nucleic acid and gRNA. The experimental results show that Cas-sf0005 can be used for the cleavage and editing of double-stranded target nucleic acid.

Example 4. Editing efficiency of Cas-sf0005 protein in animal cells

The gene editing activity of Cas-sf0005 protein was verified in animal cells, and the target was designed for the FUT8 gene of Chinese hamster ovary cells (CHO). The vector pcDNA3.3 was transformed with EGFP fluorescent protein and PuroR resistance gene. The SV40 NLS-Cas-sf0005 fusion protein was inserted through the enzyme cleavage site BsmB1; the U6 promoter and gRNA sequence were inserted through the enzyme cleavage site Mfe1. The CMV promoter promotes the expression of fusion protein SV40 NLS-Cas-sf0005-NLS-GFP. The protein Cas-sf0005-NLS and the protein GFP were linked with the linker peptide T2A. The promoter EF-1α promotes the expression of the puromycin resistance gene.

Plating: CHO cells are plated to 70-80% confluence, and the number of cells seeded in a 12-well plate is 8*10^4 cells/well.

Transfection: Plate 6-8h for transfection, add 3.25µl Lipo3000 to 125µl opti-MEM and mix well; add 3ug plasmid and 10µl P3000 to 125µllopti-MEM, and mix well. The diluted Lipo3000 was mixed with the diluted plasmid evenly, and incubated at room temperature for 5 min. The incubated mixture was added to the medium plated with cells for transfection.

Screening by adding puromycin: 24h after transfection, adding puromycin, the final concentration is 10ng/ml. Puromycin treatment for 24h was replaced with normal medium and continued to culture for 24h.

DNA was extracted, PCR amplified near the editing region, and sent to hiTOM for sequencing: cells were digested with trypsin and collected, and genomic DNA was extracted by cell/tissue genomic DNA extraction kit (Biotech). Genomic DNA was primed NAFUT8-JC-HITOM-1F1: GGAGTGAGTACGGTGTGCTCCTCCTTACTTACCCTT

GG;NAFUT8-JC-HITOM-1R1:GAGTTGGATGCTGGATGGTTGTTCTTTGGTGGGGACTA

TG amplifies the region near the target. PCR products were subjected to hiTOM sequencing.

Sequencing data analysis, the types and proportions of sequences within 15nt upstream and 10nt downstream of the target position were counted, and the sequences with SNV frequency greater than/equal to 1% or non-SNV mutation frequency greater than/equal to 0.1% were counted, and the Cas-sf0005 protein was obtained. Editing efficiency for target positions.

CHO cell FUT8 gene target sequence: gR1-FUT8: TTT GACAAACTGGGATACCCACCACA , the italic part is the PAM sequence, and the underlined area is the target region. The gRNA sequence is CCGUCAACGUUCAACGCUUGCUCGGUUCGCCGAGAC G ACAAACUGGGAUACCCACCACA , and the underlined region is the targeting region.

The analysis results showed that the editing efficiency of Cas-sf0005 in the target gR1-FUT8 of CHO cells reached 22.49%, and the editing types were all InDel. The partial sequencing results of the edited target nucleic acid are shown in Figure 3.

Although specific embodiments of the present invention have been described in detail, those skilled in the art will appreciate that various modifications and changes can be made to the details in light of all the teachings that have been published, and that these changes are all within the scope of the present invention .

SEQUENCE LISTING

<110> Shandong Shunfeng Biotechnology Co., Ltd.

<120> Novel CRISPR enzymes and systems and applications

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<170> PatentIn version 3.5

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Met Thr Pro Lys Thr Glu Thr Pro Val Gly Ala Leu Ile Lys Lys Phe

1 5 10 15

Phe Pro Gly Lys Arg Phe Gln Lys Asn Tyr Leu Lys Asp Ala Gly Lys

20 25 30

Lys Leu Lys Arg Glu Gly Glu Ala Ala Ala Val Glu Tyr Leu Ser Gly

35 40 45

Lys Gln Glu Asp His Pro Ala Asn Phe Cys Pro Pro Ala Lys Val Asn

50 55 60

Ile Leu Ala Gln Ser Arg Pro Leu Ser Glu Trp Pro Ile Asn Leu Val

65 70 75 80

Ser Lys Gly Val Gln Glu Tyr Val Tyr Gly Leu Thr Ala Ala Glu Arg

85 90 95

Glu Ala Asn Gly Asp Phe Gly Thr Ser Arg Lys Ser Leu Asp Arg Trp

100 105 110

Phe Ala Arg Thr Gly Val Pro Thr His Gly Tyr Thr Thr Val Gln Gly

115 120 125

Leu Asn Leu Ile Leu Arg His Thr Phe Asn Arg Tyr Asp Gly Val Ile

130 135 140

Lys Lys Val Glu Thr Arg Asn Glu Lys Arg Arg Ser Lys Ala Thr Arg

145 150 155 160

Ile Asn Val Ser Arg Glu Ala Asp Gly Leu Pro Pro Ile Glu Ala Glu

165 170 175

Pro Glu Glu Thr Ala Phe Gly Pro Asp Gly Lys Leu Lys Glu Arg Pro

180 185 190

Gly Ile Asn Pro Ser Ile Tyr Cys Tyr Gln Gln Val Ser Pro Val Pro

195 200 205

Tyr Asn Pro Ala Lys His Pro Ala Leu Pro Phe Ser Gly Val Asp Pro

210 215 220

Gly Ala Pro Leu Pro Leu Gly Thr Pro Asn Arg Leu Ser Ile Pro Lys

225 230 235 240

Gly Gln Pro Gly Tyr Val Pro Glu Trp Gln Arg Pro His Leu Ser Thr

245 250 255

Lys Asn Lys Arg Ile Arg Lys Trp Tyr Ala Arg Ala Asn Trp Arg Arg

260 265 270

Lys Pro Gly Arg Lys Ser Val Leu Asp Glu Ala Lys Leu Lys Glu Ala

275 280 285

Ala Leu Lys Glu Ala Ile Pro Ile Ile Val Thr Ile Gly Lys Asp Trp

290 295 300

Ile Val Met Asp Ala Arg Gly Leu Leu Arg Ala Val Tyr Trp Arg Gly

305 310 315 320

Ile Ala Lys Pro Gly Leu Ser Leu Lys Glu Leu Leu Gly Phe Phe Ser

325 330 335

Gly Asp Pro Val Leu Asp Pro Lys Arg Gly Ile Ala Thr Phe Thr Phe

340 345 350

Lys Leu Gly Ala Val Ala Val His Ser Arg Lys Pro Thr Arg Gly Lys

355 360 365

Lys Ser Lys Glu Leu Leu Leu Ser Met Thr Ala Glu Lys Pro His Val

370 375 380

Gly Leu Val Ala Ile Asp Leu Gly Gln Thr Asn Pro Val Ala Ala Glu

385 390 395 400

Phe Ser Arg Val Lys Arg Glu Gly Glu Thr Leu Gln Ala Glu Pro Leu

405 410 415

Gly Gln Ile Val Leu Pro Asp Asp Leu Val Lys Asp Leu Thr Arg Tyr

420 425 430

Arg Arg Ala Trp Asp Ala Thr Glu Glu Gln Ile Lys Ala Glu Ala Ile

435 440 445

Val Gln Leu Pro Glu Glu Cys Arg Ala Glu Val Val Lys Val Asn Gln

450 455 460

Met Ser Ala Glu Glu Thr Lys His Leu Ile Leu Asp Arg Gly Val Ser

465 470 475 480

Gly Asp Leu Pro Trp Glu Lys Met Thr Ser Asn Thr Thr Phe Ile Ser

485 490 495

Asp His Leu Leu Ala Lys Gly Val Thr Asp Gln Val Phe Phe Glu Lys

500 505 510

Lys Ser Lys Gly Lys Lys Lys Lys Gly Thr Glu Thr Val Lys Arg Lys Asp

515 520 525

Tyr Gly Trp Val Lys Leu Leu Arg Pro Arg Leu Ser Gln Glu Thr Arg

530 535 540

Lys Ala Val Asn Asp Lys Thr Trp Glu Leu Lys Arg Ala Ser Thr Glu

545 550 555 560

Tyr Val Arg Leu Ser Arg Arg Lys Thr Glu Leu Ala Arg Arg Cys Val

565 570 575

Asn Tyr Ile Val Arg Glu Thr Lys Arg Trp Thr Gln Cys Glu Asp Ile

580 585 590

Ala Ile Val Ile Glu Asp Leu Asn Val Arg Phe Phe His Gly Ser Gly

595 600 605

Glu Arg Pro Asp Gly Trp Asp Asn Phe Phe Ile Ser Lys Arg Glu Asn

610 615 620

Arg Trp Phe Ile Gln Val Leu His Lys Ala Phe Ser Asp Leu Ala Leu

625 630 635 640

His Arg Gly Leu Pro Val Ile Glu Ala Asn Pro Ala Arg Thr Ser Ile

645 650 655

Thr Cys Ile Arg Cys Gly His Cys Asp Arg Asn Asn Arg His Gly Glu

660 665 670

Met Phe Leu Cys Leu Ser Cys Asn Asp Leu Arg His Ala Asp Arg Glu

675 680 685

Ile Ala Thr Arg Asn Leu Thr Arg Val Ala Val Thr Gly Glu Met Ile

690 695 700

Pro Arg Arg Ile Glu Pro Gly Glu Gln Ser Gly Asp Thr Lys Lys Ala

705 710 715 720

Arg Ser Ala Arg Lys Gly Lys Lys Ala Val Ile Ser Lys Arg Glu Ala

725 730 735

Ala

<210> 2

<211> 2214

<212> DNA

<213> Artificial Sequence

<220>

<223> Cas-sf0005

<400> 2

atgaccccca aaaccgaaac gccggtgggc gccctcatca agaagttctt ccctggtaag 60

cggttccaga agaactacct gaaggacgca gggaagaagc tgaagaggga gggtgaggct 120

gccgctgtgg agtacctgtc ggggaagcaa gaggaccacc ccgcgaactt ctgccctcct 180

gcgaaggtca acatccttgc gcagagccgc cccctctcgg agtggcccat caacctcgtc 240

tcgaagggcg tgcaggagta cgtctacggg ctcacggccg ctgaacgtga ggcaaacggg 300

gatttcggga cctcccgtaa gagcctcgat cggtggttcg ctcgaacggg cgttcctacc 360

cacgggtaca cgaccgttca agggttgaac ctcatcctgc ggcacacctt caaccgttac 420

gacggcgtga tcaagaaggt cgagacgcgc aacgagaagc gtcgaagcaa ggccacccgc 480

atcaacgtgt cacgggaagc tgacggcctt ccgcctatcg aggcggaacc cgaagaaacg 540

gccttcggcc cggatgggaa gctgaaggag cgccctggga tcaacccctc gatctactgc 600

taccaacagg tgtcgccggt cccttacaac ccggcgaagc atcctgcgct gccttttttct 660

ggcgtcgatc ccggtgcacc tttgcccttg ggcaccccga accgcctgag tattcccaag 720

gggcagccgg ggtacgtccc ggagtggcag cggccccatc tctcgacgaa gaacaagcgt 780

atccgcaagt ggtatgcccg cgccaactgg cgccgtaagc cggggcgcaa gtctgttctg 840

gacgaggcga agctgaagga ggctgccctg aaggaggcga tccccatcat cgtcaccatc 900

ggcaaggatt ggattgtcat ggatgctcgc ggcttgctgc gggctgtcta ctggcgtggc 960

atcgcaaagc cgggcctgtc gctcaaggag cttcttggct tcttctccgg cgaccctgtt 1020

ctcgacccga agcgcggcat cgcaaccttc accttcaagc tgggcgcggt cgcagttcac 1080

tcgcgcaagc cgacgcgggg caagaagtcg aaggagctgc tcctcagcat gactgccgag 1140

aagccgcacg tgggtctggt cgccatcgac cttggacaga cgaaccctgt cgctgcggag 1200

ttctcccgtg tcaagcgcga gggagagacg ttgcaagcgg agcccctcgg gcagatcgtg 1260

ctgccggacg acctggtcaa ggatctcacg cgctatcggc gcgcatggga tgcgaccgag 1320

gagcagatca aggcggaagc catcgtgcag ctccccgaag agtgccgggc cgaggttgtg 1380

aaggtcaatc aaatgtcggc ggaagagacc aagcacctca tcctcgatag gggcgttagt 1440

ggggatctcc cctgggagaa gatgacctcg aacacgacgt tcatctccga ccacttgctc 1500

gccaagggtg tcaccgacca ggtcttcttc gagaagaaga gcaagggcaa gaagaagggc 1560

acggagacgg tcaagcgcaa ggactacgga tgggtgaagc tccttcgccc ccgtttgtcc 1620

caggagactc gcaaggctgt caacgacaag acctgggagt tgaagcgggc cagcacggag 1680

tacgtgcgtc tctcacgccg aaagactgaa ctcgcccgcc gctgtgtcaa ctacatcgtc 1740

cgagagacca agcgctggac gcagtgcgag gacatcgcca tcgtcatcga agatctcaac 1800

gtccgcttct tccacgggtc gggcgagcgc ccggatggat gggacaactt cttcatctcg 1860

aagcgcgaga accgctggtt catccaggta ctacacaagg cattcagtga cctcgccttg 1920

caccgcgggc tgccggtcat cgaggccaac cccgcaagga cgagcatcac atgcatccgg 1980

tgcgggcact gtgatcggaa taaccgccac ggggagatgt tcctgtgcct ctcgtgcaac 2040

gatctacgtc acgctgaccg tgagatcgct acccggaacc tgacccgcgt ggcggtcacg 2100

ggtgagatga taccccggcg catcgagccc ggcgagcagt cgggtgacac caaaaaggcc 2160

aggagtgctc gcaagggcaa aaaggcagtg atttcgaaga gggaggctgc ttag 2214

<210> 3

<211> 2214

<212> DNA

<213> Artificial Sequence

<220>

<223> Cas-sf0005-hu

<400> 3

atgacaccca aaaccgagac acctgtgggc gctctgatta agaaattctt ccccggcaag 60

agattccaga agaactacct gaaggacgcc ggcaagaagc tgaagagaga gggcgaggct 120

gctgccgttg aatatttaag cggaaaacaa gaagatcacc ccgccaactt ctgccccccc 180

gctaaagtga atatcctggc ccaaagcaga cccctgagcg agtggcctat taatctggtg 240

agcaagggcg tgcaggagta cgtgtatggc ctgaccgctg ccgaaagaga ggctaatggc 300

gatttcggaa ccagcagaaa gagcctggac agatggttcg ccagaaccgg cgtgcctacc 360

catggatata caacagtgca aggcctgaac ctgattctga gacacacctt caacagatac 420

gacggcgtga tcaagaaggt ggaaaccaga aacgagaaga gaagaagcaa ggccaccaga 480

atcaacgtga gcagagaggc cgacggcctg cctcctattg aagccgaacc tgaagagaca 540

gcttttggcc ccgatggaaa actgaaagag aggcccggca tcaaccccag catctactgc 600

taccagcagg tgagccccgt gccctataat cccgctaagc atcctgctct gcccttcagc 660

ggagtggacc ccggagctcc tctgcctctg ggaacaccta atagactgag cattcccaag 720

ggccagcccg gctatgttcc tgaatggcaa agaccccacc tgagcacaaa gaacaagaga 780

atcagaaagt ggtacgccag agccaactgg agaagaaagc ccggcagaaa gagcgtgctg 840

gacgaggcta aactgaagga ggccgctctg aaagaggcca tccctattat cgtgaccatc 900

ggcaaggact ggatcgtgat ggacgccaga ggcctgctga gagccgtgta ttggagaggc 960

attgccaagc ccggactgag cctgaaggaa ctgctgggat ttttcagcgg cgatcctgtg 1020

ctggatccta agagaggcat tgccaccttc acctttaagc tgggcgccgt ggctgtgcac 1080

agcagaaaac ctacaagagg caagaagagc aaggagctgc tgctgagcat gaccgccgaa 1140

aaaccccacg tgggcctggt ggctattgat ctgggccaaa caaatcctgt ggccgctgaa 1200

ttcagcagag tgaaaagaga gggcgagaca ctgcaggccg aacccttagg acaaattgtg 1260

ctgcctgatg atctggtgaa ggacctgacc agatacagaa gagcctggga cgccaccgag 1320

gagcaaatta aagccgaagc tatcgtgcag ctgcccgaag agtgtagagc cgaggtggtg 1380

aaagtgaacc agatgagcgc cgaggagaca aagcacctga ttctggatag aggcgtgagc 1440

ggcgatctgc cttgggaaaa aatgaccagc aacaccacct tcatcagcga ccacctgctg 1500

gccaaaggcg tgacagatca agtgtttttc gagaagaaga gcaagggcaa gaagaagggc 1560

accgaaaccg tgaagagaaa ggactacggc tgggtgaagc tgctcagacc cagactgagc 1620

caggagacaa gaaaggccgt gaacgacaag acctgggagc tgaagagagc cagcaccgag 1680

tatgtgagac tgagcagaag aaagaccgag ctggccagaa gatgcgtgaa ctacatcgtg 1740

agagagacaa agagatggac ccagtgcgag gacatcgcca tcgtgattga ggacctgaac 1800

gtgagattct tccacggcag cggcgaaaga cccgatggat gggataattt cttcatcagc 1860

aagagagaga acagatggtt catccaggtg ctgcacaagg ccttcagcga cctggctctg 1920

catagaggac tgcctgtgat tgaagccaat cctgccagaa caagcatcac ctgtatcaga 1980

tgcggccact gcgacagaaa caacagacac ggcgagatgt tcctgtgcct gagctgcaat 2040

gacctgagac acgccgacag agagatcgcc acaagaaacc tgaccagagt ggccgtgacc 2100

ggcgaaatga ttcccagaag aatcgagccc ggcgagcaga gcggagatac aaaaaaagct 2160

agaagcgcca gaaagggcaa gaaggccgtg atcagcaaga gagaggccgc ctaa 2214

<210> 4

<211> 36

<212> RNA

<213> Artificial Sequence

<220>

<223> DR

<400> 4

ccgucaacgu ucaacgcuug cucgguucgc cgagac 36

<210> 5

<211> 36

<212> RNA

<213> Artificial Sequence

<220>

<223> DR

<400> 5

ccgucaacgu ucaacgcuug cucgguacgc cgagac 36

Claims (24)

1. A Cas protein, characterized in that the amino acid sequence of the Cas protein is shown in SEQ ID No.1. 2. A fusion protein comprising the Cas protein of claim 1 and other modified parts. 3. An isolated polynucleotide, wherein the polynucleotide is a polynucleotide sequence encoding the Cas protein of claim 1, or a polynucleotide sequence encoding the fusion protein of claim 2. 4. A vector, characterized in that the vector comprises the polynucleotide of claim 3 and a regulatory element operably linked thereto. 5. A CRISPR-Cas system, wherein the system comprises the Cas protein of claim 1 and at least one gRNA. 6. A carrier system, characterized in that the carrier system comprises one or more carriers, the one or more carriers comprising: a) a first regulatory element operably linked to the gRNA, b) a second regulatory element operably linked to the Cas protein of claim 1; wherein components (a) and (b) are on the same or different supports of the system. 7. A composition, characterized in that the composition comprises: (i) protein component, it is selected from: the described Cas protein of claim 1 or the described fusion protein of claim 2; (ii) a nucleic acid component, which is a gRNA; The protein component and the nucleic acid component combine with each other to form a complex. 8. An activated CRISPR complex comprising: (i) protein component, it is selected from: the Cas protein of claim 1 or the fusion protein of claim 2; (ii) a nucleic acid component, which is a gRNA; (iii) a target sequence that binds to the gRNA described in (ii). 9. An engineered host cell, 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 4. The vector of claim 4, wherein the host cell is a cell of a species other than an animal or plant. 10. 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, 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 editing Applications, which are applications for non-disease diagnosis and treatment purposes. The application according to claim 10, wherein the gene editing is gene targeting or gene cutting. 12. 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- The Cas system, or the vector system according to claim 6, or the composition according to claim 7, or the activated CRISPR complex according to claim 8, or the host cell according to claim 9 are used in the preparation of gene Edited reagents or kits for use. 13. The use according to claim 12, wherein the gene editing is gene targeting or gene cutting. 14. 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- The Cas system, 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 editing a target nucleic acid The application of the application is not for the purpose of disease diagnosis and treatment. 15. The use according to claim 14, wherein the editing target nucleic acid is selected from cutting double-stranded DNA, single-stranded DNA or single-stranded RNA, or targeting target nucleic acid. 16. The use according to claim 14, wherein the editing target nucleic acid is selected from base-edited single-stranded nucleic acid or base-edited double-stranded nucleic acid. 17. 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, 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 preparation for editing Use of reagents or kits for target nucleic acids. 18. The use according to claim 17, wherein the editing target nucleic acid is selected from cutting double-stranded DNA, single-stranded DNA or single-stranded RNA, or targeting target nucleic acid. 19. The use according to claim 17, wherein the editing target nucleic acid is selected from base-edited single-stranded nucleic acid or base-edited double-stranded nucleic acid. 20. A method for editing a target nucleic acid, which is a method for non-disease diagnosis and treatment purposes, the method comprising combining 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 combination of claim 7 contact with the substance, or the activated CRISPR complex of claim 8, or the host cell of claim 9. 21. The method according to claim 20, wherein the editing target nucleic acid is a targeting target nucleic acid or a cleaving target nucleic acid. 22. A kit for gene editing, the kit comprising the Cas protein of claim 1, or the fusion protein of claim 2, or the polynucleotide of claim 3, or the claim The vector of 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. 23. 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- The Cas system, 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 are preparing a preparation or reagent Use in a kit, the formulation or kit for: (i) genome editing; (ii) target nucleic acid detection; (iii) Treatment of disease. 24. The use of claim 23, wherein the genome editing modifies an organism by editing a target sequence in a target locus.

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