WO2022166895A1 - Crispr酶和系统以及应用 - Google Patents

Crispr酶和系统以及应用 Download PDF

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WO2022166895A1
WO2022166895A1 PCT/CN2022/075030 CN2022075030W WO2022166895A1 WO 2022166895 A1 WO2022166895 A1 WO 2022166895A1 CN 2022075030 W CN2022075030 W CN 2022075030W WO 2022166895 A1 WO2022166895 A1 WO 2022166895A1
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nucleic acid
protein
cas
grna
sequence
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French (fr)
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李珊珊
梁亚峰
赵庆芝
孙洁
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山东舜丰生物科技有限公司
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    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]

Definitions

  • the present invention relates to the field of gene editing, in particular to the technical field of regularly clustered interspaced short palindromic repeats (CRISPR).
  • CRISPR regularly clustered interspaced short palindromic repeats
  • the present invention relates to CRISPR enzymes and systems and applications, in particular 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.
  • 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.
  • the CRISPR/Cas Type V system is a newly discovered CRISPR system, which has a 5'-TTN motif for sticky end cleavage of target sequences, such as Cpf1, C2c1, CasX, CasY.
  • target sequences such as Cpf1, C2c1, CasX, CasY.
  • the different CRISPR/Cas that currently exist have different advantages and disadvantages.
  • 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.
  • 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.
  • the inventors of the present application have unexpectedly discovered a novel endonuclease (Cas enzyme) after a lot of experiments and repeated explorations. Based on this discovery, the present inventors developed a new CRISPR/Cas system and gene editing methods and nucleic acid detection methods based on the system.
  • the present invention provides a Cas protein, which is an effector protein in the CRISPR/Cas system, and is also referred to as a Cas-sf19 protein in the present invention.
  • 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 SEQ ID NO: 1.
  • the Cas protein of the present invention and the Cas-sf19 protein are derived from the same species.
  • the Cas protein amino acid sequence has one or more amino acid substitutions, deletions or additions compared to SEQ ID NO: 1; the one or more amino acids include 1, 2, Substitution, deletion or addition of 3, 4, 5, 6, 7, 8, 9 or 10 amino acids.
  • the Cas protein of the present invention has any one or any of several functional domains in the following i-iii:
  • 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.
  • 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.
  • the protein of the present invention may comprise one or more conservative substitutions in the amino acid sequence, and these conservative substitutions are preferably produced by substitution according to Table 1.
  • 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.
  • 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.
  • Constant 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 non-conserved regions of Cas-sf19. 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.
  • 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.
  • amino acid sequence variants of the Cas-sf19 protein 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.
  • these minor amino acid changes in the Cas-sf19 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 Cas-sf19 protein 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.
  • the Cas protein contains the amino acid sequence shown in SEQ ID NO: 1.
  • the Cas protein is the amino acid sequence shown in SEQ ID NO: 1.
  • 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 with 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 and Trans cleavage active.
  • the present invention also provides a fusion protein comprising the above-mentioned Cas protein and other modified parts.
  • the modified moiety is selected from an additional protein or polypeptide, a detectable label, or any combination thereof.
  • 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 (e.g., 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 random combination.
  • NLS sequences are well known to those skilled in the art, examples of which include, but are not limited to, the SV40
  • the NLS sequence is located at, near or near the terminus (eg, N-terminus, C-terminus or both) of the Cas protein of the invention.
  • 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).
  • 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.
  • 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.
  • MBP maltose binding protein
  • DBD DNA binding domain of Lex A
  • GAL4 GAL4
  • the fusion proteins of the present invention comprise a detectable label, such as a fluorescent dye, such as FITC or DAPI.
  • the Cas protein of the invention is coupled, conjugated or fused to the modified moiety, optionally via a linker.
  • the modified moiety is directly attached to the N-terminus or C-terminus of the Cas protein of the invention.
  • the modified moiety is linked to the N-terminus or C-terminus of the Cas protein of the invention via a linker.
  • 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, ⁇ -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 method (recombinant technology) or chemical synthesis method.
  • the present invention provides an isolated polynucleotide comprising:
  • the homology between the nucleotide sequence and the sequence shown in SEQ ID NO: 2 is ⁇ 80% (preferably ⁇ 90%, more preferably ⁇ 95%, optimally ⁇ 98%), and encodes SEQ ID
  • the polynucleotide of the polypeptide shown in NO: 1; or, the homology between the nucleotide sequence and the sequence shown in SEQ ID NO: 2 is ⁇ 80% (preferably ⁇ 90%, more preferably ⁇ 95%, the best ⁇ 98%), and the polynucleotide encoding the polypeptide shown in SEQ ID NO: 1; or, the homology between the nucleotide sequence and the sequence shown in SEQ ID NO: 2 is ⁇ 80% (preferably ⁇ 90%) , more preferably ⁇ 95%, optimally ⁇ 98%), and the polynucleotide encoding the polypeptide shown in SEQ ID NO: 1; or,
  • nucleotide sequence of any 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.
  • the polynucleotide is preferably single-stranded or double-stranded.
  • the present invention provides an engineered direct repeat sequence that forms a complex with the above Cas protein.
  • the direct repeat sequence is connected with a guide sequence capable of hybridizing with the target sequence to form a guide RNA (guide RNA or gRNA).
  • guide RNA or gRNA guide RNA
  • the hybridization of the target sequence and the gRNA representing at least 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% identity so that they can hybridize to form a complex; or at least 12, 15, 16, 17, 18 nucleic acid sequences representing the target sequence and the gRNA, 19, 20, 21, 22, or more bases can complementarily pair to form a complex.
  • the direct repeat has at least 90% sequence identity to SEQ ID NO:3. In some embodiments, the direct repeat sequence has one or more base substitutions, deletions or additions (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 base substitutions, deletions or additions).
  • the direct repeat is shown in SEQ ID NO:3.
  • gRNA guide RNA
  • 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 “Direct Repeat (Direct Repeat) sequence”; the second segment is also referred to as “targeting sequence targeting nucleic acid” or “targeting segment targeting nucleic acid”, or “targeting target” Sequence's Guide 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.
  • 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.
  • 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).
  • 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.
  • the guide RNA comprises a first segment and a second segment from the 5' to 3' direction.
  • 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 protein of the present invention comprises a guide sequence hybridized with a target sequence, wherein the target sequence is located at the 3' end of the protospacer adjacent motif (PAM), and the PAM sequence is 5'-ATG-3' .
  • PAM protospacer adjacent motif
  • 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.
  • the regulatory elements are selected from one or more of the group consisting of enhancers, transposons, promoters, terminators, leader sequences, polyadenylation sequences, marker genes.
  • the vectors include cloning vectors, expression vectors, shuttle vectors, and integration vectors.
  • 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.
  • viral vectors eg, retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated vectors and herpes simplex vectors
  • 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.
  • the nucleic acid sequence encoding the Cas protein and the nucleic acid encoding one or more guide RNAs are artificially synthesized.
  • 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.
  • the Cas protein is codon-optimized for expression in a cell.
  • 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:
  • 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 or inducible promoters), enhancers (e.g., 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).
  • promoters e.g., constitutive or inducible promoters
  • enhancers e.g., 35S promoter or 35S enhanced promoter
  • IVS internal ribosome entry sites
  • Expression control elements eg, transcription termination signals such as polyadenylation signals and poly U sequences.
  • 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.
  • viral vectors eg, retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated vectors and herpes simplex vectors
  • plasmids viruses, cosmids, phages and other types, which are well known to those skilled in the art.
  • the systems provided herein are in a delivery system.
  • delivery systems are nanoparticles, liposomes, exosomes, microvesicles, and gene guns.
  • 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.
  • the target sequence when the target sequence is DNA, the target sequence is located at the 3' end of the protospacer adjacent motif (PAM), and the PAM has a sequence shown in 5'-ATG-3' .
  • PAM protospacer adjacent motif
  • 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.
  • the Cas protein is linked to one or more NLS sequences.
  • the fusion protein comprises one or more NLS sequences.
  • the NLS sequence is linked to the N-terminus or the C-terminus of the protein.
  • the NLS sequence is fused to the N-terminus or C-terminus of the protein.
  • the present invention relates to an engineered CRISPR system comprising the above-mentioned 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.
  • Protein-nucleic acid complexes/compositions Protein-nucleic acid complexes/compositions
  • the present invention provides a composite or composition comprising:
  • a protein component selected from the group consisting of the above Cas proteins, derivatized proteins or fusion proteins, and any combination thereof;
  • 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.
  • the nucleic acid component is a guide RNA in a CRISPR-Cas system.
  • 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.
  • 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.
  • the binding is through the targeting sequence of the targeting nucleic acid on the gRNA and the target nucleic acid.
  • activated CRISPR complex 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.
  • 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.
  • the activated CRISPR complex may exhibit side nuclease cleavage activity, and the side nuclease activity refers to the non-specific cleavage activity or disorder of single-stranded nucleic acid exhibited by the activated CRISPR complex.
  • cleavage activity also known as trans cleavage activity in the art.
  • 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.
  • the present invention provides a delivery composition
  • 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.
  • the delivery vehicle is a particle.
  • 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).
  • viral vectors eg, replication-defective retroviruses , lentivirus, adenovirus or adeno-associated virus.
  • 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.
  • the cells are prokaryotic cells.
  • 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).
  • poultry birds eg, chickens
  • fish or crustaceans
  • 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, trees or producing plants, fruits or vegetables (eg, trees such as citrus trees, nut trees; nightshade, cotton, tobacco, tomatoes, grapes, coffee, cocoa, etc.).
  • the cells are stem cells or stem cell lines.
  • the host cells of the invention comprise a modification of a gene or genome that is not present in its wild type.
  • 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; non-specifically cutting and/or degrading side nucleic acids; non-specifically cutting single-stranded nucleic acids; nucleic acid detection; detecting nucleic acids in target samples; specifically Editing double-stranded nucleic acids; base-editing double-stranded nucleic acids; base-editing single-stranded nucleic acids. 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, 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 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.
  • 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.
  • 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.
  • the present invention also provides the application of the above-mentioned Cas protein, nucleic acid, the above-mentioned composition, the above-mentioned CIRSPR/Cas system, the above-mentioned carrier system, the above-mentioned delivery composition or the above-mentioned activated CRISPR complex in nucleic acid detection, or in the preparation of Use in reagents or kits for nucleic acid detection.
  • the present invention also provides a method for cleaving a single-stranded nucleic acid, the method comprising contacting a nucleic acid population with the above-mentioned Cas protein and gRNA, wherein the nucleic acid population comprises a target nucleic acid and a plurality of non-target single-stranded nucleic acids , the Cas protein cleaves the plurality of non-target single-stranded nucleic acids.
  • the gRNA is capable of binding the Cas protein.
  • the gRNA is capable of targeting the target nucleic acid.
  • the contacting can be inside a cell in vitro, ex vivo or in vivo.
  • the cleavage of single-stranded nucleic acid is non-specific cleavage.
  • the present invention also provides the above-mentioned Cas protein, nucleic acid, the above-mentioned composition, the above-mentioned CIRSPR/Cas system, the above-mentioned carrier system, the above-mentioned delivery composition or the above-mentioned activated CRISPR complex in non-specific cutting of single-stranded nucleic acid. application, or use in the preparation of reagents or kits for non-specific cleavage of single-stranded nucleic acids.
  • 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.
  • the present 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 that is single-stranded and does not hybridize to the guide RNA Detector.
  • precursor RNAs can be cleaved or processed into mature guide RNAs as described above.
  • 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.
  • the formulation or kit is for:
  • the above-mentioned gene or genome editing is intracellular or extracellular gene or genome editing.
  • the target nucleic acid detection and/or diagnosis is in vitro target nucleic acid detection and/or diagnosis.
  • the treatment of the disease is the treatment of a disorder caused by a defect in the target sequence in the target locus.
  • the present invention provides a method for detecting a target nucleic acid in a sample, the method comprising contacting the sample with the Cas protein, a gRNA (guide RNA) and a single-stranded nucleic acid detector, the gRNA comprising The region bound by Cas protein and the guide sequence that hybridizes to the target nucleic acid; the detectable signal generated by the cleavage of the single-stranded nucleic acid detector by the Cas protein is detected, thereby detecting the target nucleic acid; the single-stranded nucleic acid detector does not hybridize to the gRNA .
  • 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.
  • the cells are selected from prokaryotic or eukaryotic cells, eg, animal cells, plant cells, or microbial cells.
  • 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.
  • 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.
  • the modification further comprises inserting an editing template (eg, exogenous nucleic acid) into the fragment.
  • an editing template eg, exogenous nucleic acid
  • the method further comprises: contacting an editing template with the target nucleic acid, or delivering to a cell comprising the target nucleic acid.
  • 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.
  • the present invention provides a method for detecting a target nucleic acid in a sample, the method comprising combining the sample with the above-mentioned Cas protein, nucleic acid, the above-mentioned composition, the above-mentioned CIRSPR/Cas system, the above-mentioned carrier system, the above-mentioned delivery composition or
  • the above activated CRISPR complex is contacted with the single-stranded nucleic acid detector; the detectable signal generated by the Cas protein cleaving the single-stranded nucleic acid detector is detected, thereby detecting the target nucleic acid.
  • the target nucleic acid includes ribonucleotides or deoxyribonucleotides; including single-stranded nucleic acid, double-stranded nucleic acid, such as single-stranded DNA, double-stranded DNA, single-stranded RNA, double-stranded RNA.
  • the target nucleic acid is derived from samples such as viruses, bacteria, microorganisms, soil, water sources, human body, animals, plants and the like.
  • the target nucleic acid is a product enriched or amplified by methods such as PCR, NASBA, RPA, SDA, LAMP, HAD, NEAR, MDA, RCA, LCR, RAM and the like.
  • the target nucleic acid is viral nucleic acid, bacterial nucleic acid, disease-related specific nucleic acid, such as a specific mutation site or SNP site or a nucleic acid that is different from a control;
  • the virus is a plant Virus or animal virus, for example, papilloma virus, hepadnavirus, herpes virus, adenovirus, poxvirus, parvovirus, coronavirus;
  • the virus is a coronavirus, preferably SARS, SARS-CoV2 (COVID-19) -19), HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, Mers-Cov.
  • the gRNA has at least 50% matching degree with the target sequence on the target nucleic acid, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%.
  • the characteristic sites are completely matched to the gRNA.
  • one or more gRNAs with different targeting sequences may be included in the detection method, which target different target sequences.
  • the single-stranded nucleic acid detectors include but are not limited to single-stranded DNA, single-stranded RNA, DNA-RNA hybrids, nucleic acid analogs, base modifications, and single-stranded nucleic acid detections containing abasic spacers
  • Nucleic acid analogs include but are not limited to: locked nucleic acid, bridge nucleic acid, morpholine nucleic acid, glycol nucleic acid, hexitol nucleic acid, threose nucleic acid, arabinose nucleic acid, 2'oxymethyl RNA, 2' Methoxyacetyl RNA, 2' fluoro RNA, 2' amino RNA, 4' thio RNA, and combinations thereof, including optional ribonucleotide or deoxyribonucleotide residues.
  • the detectable signal is realized by the following methods: vision-based detection, sensor-based detection, color detection, fluorescence signal-based detection, gold nanoparticle-based detection, fluorescence polarization, colloidal phase transition/dispersion, electrical Chemical detection and semiconductor-based detection.
  • two ends of the single-stranded nucleic acid detector are respectively provided with a fluorescent group and a quenching group, and when the single-stranded nucleic acid detector is cleaved, a detectable fluorescent signal can be displayed.
  • the fluorescent group is selected from one or any of FAM, FITC, VIC, JOE, TET, CY3, CY5, ROX, Texas Red or LC RED460;
  • the quenching group is selected from BHQ1, BHQ2, BHQ3 , one or any of Dabcy1 or Tamra.
  • the 5' end and the 3' end of the single-stranded nucleic acid detector are respectively provided with different label molecules, and the detection of the single-stranded nucleic acid detector before and after being cleaved by Cas protein is detected by colloidal gold detection.
  • the method of detecting a target nucleic acid can 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.
  • the method of detecting a target nucleic acid can also include using RNA reporter nucleic acid and DNA reporter nucleic acid (eg, fluorescent colors) on separate channels, and by measuring the signal levels of the RNA and DNA reporter molecules, and by measuring The amount of target nucleic acid in the RNA and DNA reporter molecules determines the level of detectable signal, sampling based on combining (eg, using a minimum or product) the levels of the detectable signal.
  • RNA reporter nucleic acid and DNA reporter nucleic acid eg, fluorescent colors
  • the target gene is present in a cell.
  • the cells are prokaryotic cells.
  • the cell is a eukaryotic cell.
  • the cells are animal cells.
  • the cells are human cells.
  • the cells are plant cells, such as cells possessed by cultivated plants (eg, cassava, corn, sorghum, wheat, or rice), algae, trees, or vegetables.
  • cultivated plants eg, cassava, corn, sorghum, wheat, or rice
  • algae e.g., trees, or vegetables.
  • the target gene is present in a nucleic acid molecule (eg, a plasmid) in vitro.
  • a nucleic acid molecule eg, a plasmid
  • the target gene is present in a plasmid.
  • 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:
  • amino acid substitutions, deletions or additions e.g. 1, 2, 3, 4, 5, 6, 7, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions
  • Nucleic acid cleavage or cleavage of nucleic acids herein includes: DNA or RNA breaks in target nucleic acids (Cis cleavage), DNA or RNA breaks in collateral nucleic acid substrates (single-stranded nucleic acid substrates) ( i.e. non-specific or non-targeted, Trans cleavage).
  • the cleavage is a double-stranded DNA break.
  • the cleavage is a single-stranded DNA break or a single-stranded RNA break.
  • CRISPR-Cas system CRISPR-associated (Cas)
  • CRISPR system CRISPR system
  • 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 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
  • 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.
  • 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.
  • the degree of complementarity between a 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 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.
  • the target sequence is intracellular or extracellular.
  • the target sequence is located in the nucleus or cytoplasm of the cell.
  • 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.”
  • the editing template is an exogenous nucleic acid.
  • the recombination is homologous recombination.
  • a "target sequence” or “target polynucleotide” or “target nucleic acid” may be any polynucleotide, either endogenous or exogenous to a cell (eg, a eukaryotic cell).
  • 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).
  • the target sequence should be associated with a protospacer adjacent motif (PAM).
  • PAM protospacer adjacent motif
  • the single-stranded nucleic acid detector described in the present invention refers to a sequence containing 2-200 nucleotides, preferably, 2-150 nucleotides, preferably, 3-100 nucleotides, preferably, 3-30 nucleotides Nucleotides, preferably, 4-20 nucleotides, more preferably, 5-15 nucleotides.
  • Preferred are single-stranded DNA molecules, single-stranded RNA molecules or single-stranded DNA-RNA hybrids.
  • the two ends of the single-stranded nucleic acid detector include different reporter groups or label molecules. When it is in an initial state (ie, when it is not in a cleaved state), it does not present a report signal. When the single-stranded nucleic acid detector is cleaved, it presents A detectable signal is detected, that is, a detectable difference is exhibited after cleavage and before cleavage.
  • the reporter group or labeling molecule includes a fluorescent group and a quenching group
  • the fluorescent group is selected from FAM, FITC, VIC, JOE, TET, CY3, CY5, ROX, Texas Red Or one or any of LC RED460
  • the quenching group is selected from one or any of BHQ1, BHQ2, BHQ3, Dabcyl or Tamra.
  • the single-stranded nucleic acid detector has a first molecule (such as FAM or FITC) attached to the 5' end and a second molecule (such as biotin) attached to the 3' end.
  • the reaction system containing the single-stranded nucleic acid detector cooperates with the flow strip to detect target nucleic acid (preferably, colloidal gold detection method).
  • the flow strip is designed to have two capture lines, an antibody that binds to the first molecule (ie, the first molecule antibody) is provided at the sample contact end (colloidal gold), and the first line (control line) contains the first molecule that binds to the first molecule.
  • an antibody of a molecule of antibody that contains a second molecule of antibody bound to a second molecule at the test line ie, a second molecule of antibody, such as avidin.
  • the first molecule of antibody binds to the first molecule carrying cleaved or uncleaved oligonucleotides to the capture line, where the cleaved reporter will bind to the first molecule of antibody at the first capture line antibody, while the uncleaved reporter will bind the second molecule of antibody at the second capture line. Binding of the reporter group at each line will result in a strong readout/signal (eg color).
  • the present invention relates to the use of a flow strip as described herein for the detection of nucleic acids.
  • the invention relates to a method for the detection of nucleic acids using a flow strip as defined herein, such as a (lateral) flow assay or a (lateral) flow immunochromatographic assay.
  • the molecules in the single-stranded nucleic acid detector can be replaced with each other, or the positions of the molecules can be changed, as long as the reporting principle is the same as or similar to the present invention, the improved methods are also included in the present invention.
  • the detection method of the present 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 strength of the reporter group, for example, according to the luminescence intensity of the fluorophore, or according to the width of the color band.
  • 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.
  • 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, the 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 to say, the derivative of the protein has the same activity as the protein.
  • 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.
  • nucleic acid molecule or polypeptide 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.
  • 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 is used to refer to the match of sequences between two polypeptides or between two nucleic acids.
  • 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)
  • 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.
  • the DNA sequences CTGACT and CAGGTT share 50% identity (matching at 3 positions out of a total of 6).
  • 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.).
  • Align program DNAstar, Inc.
  • Appl Biosci., 4:11-17 (1988)) integrated 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 to determine the percent identity between two amino acid sequences.
  • 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 .
  • 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.
  • the vector may also contain an origin of replication site.
  • Plasmid refers to a circular double-stranded DNA loop into which additional DNA fragments can be inserted, eg, by standard molecular cloning techniques.
  • 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.
  • viruses eg, retroviruses, replication-defective retroviruses, adenoviruses, replication-defective adenoviruses, and adeno-associated virus
  • 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
  • vectors eg, non-episomal mammalian vectors
  • 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”.
  • 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.
  • 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 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 , California (1990).
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • NLSs typically 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.
  • the NLS comprises the PKKKRKV sequence.
  • the NLS comprises the AVKRPAATKKAGQAKKKKLD sequence.
  • the NLS comprises the PAAKRVKLD sequence.
  • 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 Mat ⁇ 2, and PY-NLS.
  • 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 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 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 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.
  • a nucleic acid eg, RNA, DNA
  • 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).
  • sequence of a polynucleotide need not be 100% complementary to the sequence of its target nucleic acid to hybridize specifically.
  • Polynucleotides 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, 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 One, 21, 22 or more bases can complementarily pair and hybridize to form a complex.
  • 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.
  • 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, eg, Holliger, P. et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R.J. et al. (1994) Structure 2:1121- 1123).
  • treating refers to treating or curing a disorder, delaying the onset of symptoms of the disorder, and/or slowing the progression of the disorder.
  • the term "subject” includes, but is not limited to, various animals, plants and microorganisms.
  • mammals such as bovine, equine, ovine, porcine, canine, feline, lagomorph, rodent (eg, mouse or rat), non-human primate an animal (eg, a macaque or cynomolgus monkey) or a human.
  • the subject eg, human
  • has a disorder eg, a disorder caused by a disease-related gene defect.
  • 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
  • Cas-sf19 is used for the cleavage results of double-stranded nucleic acids.
  • SEQ ID NO: describe 1 Amino acid sequence of Cas-sf19 2 Nucleic acid sequence of Cas-sf19 3 DR region of gRNA of Cas-sf19
  • the inventors analyzed the uncultured metagenome, and identified a new Cas enzyme through de-redundancy and protein cluster analysis. Its amino acid sequence is shown in SEQ ID NO: 1, and its nucleic acid sequence is shown in SEQ ID NO: 1. ID NO: 2. Blast results showed that the Cas protein had a low sequence identity with the reported Cas protein, and was named Cas-sf19 in the present invention.
  • the Cas-sf19 protein contains 911 amino acids, which is compared with the domains in the Pfam database.
  • the analysis results are shown in Figure 1; three functional domains are predicted, and the domain is RuvC_1 (PF18516) RuvC nuclease domain.
  • RuvC_1 PF18516
  • RuvC nuclease domain With endoribonuclease activity, the information of the three RuvC nuclease domains in the Cas-sf19 protein sequence is as follows:
  • the trans cleavage activity of Cas-sf19 was verified by in vitro assay.
  • the Cas-sf19 protein is used to guide the Cas-sf19 protein to recognize and bind to the target nucleic acid, which can be paired with the target nucleic acid; then, the Cas-sf19 protein stimulates the trans-cutting activity of any single-stranded nucleic acid, thereby cutting the single-stranded nucleic acid in the system.
  • the two ends of the single-stranded nucleic acid detector are respectively provided with a fluorescent group and a quenching group, and if the single-stranded nucleic acid detector is cut, fluorescence will be excited; in other embodiments, the two ends of the single-stranded nucleic acid detector are The ends can also be provided with labels that can be detected by colloidal gold.
  • the selected target nucleic acid is single-stranded DNA, N-B-i3g1-ssDNA0, whose sequence is: CGACATTCCGAAGAACGCTGAAGCGCTGGGGGCAAATTGTGCAATTTGCGGC;
  • the gRNA sequence is: GUGCUGCCGGUCUCUAAUCGGGGAUCGGAAUUGCAC CCCCCAGCGCUUCAGCG UUC (the underlined region is the targeting region);
  • the sequence of the single-stranded nucleic acid detector is FAM-TTGTT-BHQ1;
  • the following reaction system was used: the final concentration of Cas-sf19 was 50 nM, the final concentration of gRNA was 50 nM, the final concentration of target nucleic acid was 50 nM, and the final concentration of single-stranded nucleic acid detector was 200 nM. Incubate at 37°C and read FAM fluorescence/1min. No target nucleic acid was added to the control group.
  • Cas-sf19 protein expression plasmid The nucleic acid sequence was codon-optimized in E. coli, and then gene synthesis was performed, and then linked into the E. coli expression vector PeT28(a)+ vector.
  • the JM23119 promoter was added to the PeT28(a)+-Cas-sf19 protein vector to initiate the transcription of Cas-sf19 CrRNA.
  • Formation vector PeT28(a)+-Cas-sf19-JM23119-crRNA; construction of PAM library: synthetic sequence CGTGTTTCGTAAAGTCTGGAAACGCGGAAGCCCCAGCGCTTCAGCGTTCNNNNNNTCCCCTACGTGCTGCTGAAGTTGCCCGCAA, N is random deoxynucleotide.
  • 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.
  • PAM library subtraction experiments Preparation of competent: BL21(DE3)-PeT28(a)+-Cas-sf19-JM23119-crRNA.
  • PAM library plasmid transformation competence BL21(DE3)-PeT28(a)+-Cas-sf19-JM23119-crRNA, spread on LB plates containing kanamycin and chloramphenicol, and cultured at 37°C overnight to collect bacteria The plasmid was extracted with a large extraction kit, and the reduced PAM library was obtained.
  • control group samples were obtained by PCR reaction with 30ng/ ⁇ L plasmid (PAM library) as template primer, and the experimental group samples were obtained by PCR reaction with 30ng/ ⁇ L plasmid (PAM library after subtraction) as template.
  • the samples from the control group and the experimental group were sent to next-generation sequencing for data analysis.
  • the trans cleavage activity stimulated by Cas-sf19 targeting double-stranded DNA in vitro is verified by in vitro detection.
  • the Cas-sf19 protein is used to guide the Cas-sf19 protein to recognize and bind to the target nucleic acid, which can be paired with the target nucleic acid; then, the Cas-sf19 protein stimulates the trans-cutting activity of any single-stranded nucleic acid, thereby cutting the single-stranded nucleic acid in the system.
  • the two ends of the single-stranded nucleic acid detector are respectively provided with a fluorescent group and a quenching group, and if the single-stranded nucleic acid detector is cut, fluorescence will be excited; in other embodiments, the two ends of the single-stranded nucleic acid detector are The ends can also be provided with labels that can be detected by colloidal gold.
  • the selected target nucleic acid is double-stranded DNA (plasmid), TNB-ATG, and its sequence is: AACATTGGCCGCAAATTGCACAAT CCCCCAGCGCTTCAGCGTTC TTCGGAATGTCGCGCATTGGCATGGAAGTCACACCTTCGGGAACGTGGTTGACCTACACAGGTGCCATCAAATTGGATGACAAAGATCCAAATTTCA is linked into the vector T-Vector-pEASY-Blunt Simple Cloning Vector; the italicized part (ATG) is the PAM sequence, and the underlined area is the targeting region.
  • the gRNA sequence is: GUGCUGCCGGUCUCUAAUCGGGGAUCGGAAUUGCAC CCCCCAGCGCUUCAGCG UUC (the underlined region is the targeting region);
  • the sequence of the single-stranded nucleic acid detector is FAM-TTATT-BHQ1;
  • the following reaction system was used: the final concentration of Cas-sf19 was 100 nM, the final concentration of gRNA was 50 nM, the final concentration of double-stranded target nucleic acid was 5 ng/ ⁇ L, and the final concentration of single-stranded nucleic acid detector was 200 nM. Incubate at 37°C and read FAM fluorescence/30s.
  • This example detects the cis cleavage activity of the double-stranded DNA of Cas-sf19.
  • a gRNA that can be paired with a target nucleic acid is used to guide the Cas-sf19 protein to recognize and bind to the target nucleic acid, thereby cutting the target nucleic acid in the system, and the cleaved target nucleic acid is detected by agarose electrophoresis.
  • the selected target nucleic acid is double-stranded DNA (plasmid), TNB-ATG, and its sequence is: AACATTGGCCGCAAATTGCACAAT CCCCCAGCGCTTCAGCGTTC TTCGGAATGTCGCGCATTGGCATGGAAGT CACACCTTCGGGAACGTGGTTGACCTACACAGGTGCCATCAAATTGGATGACAAAGATCCAAATTTCA was linked into the vector T-Vector-pEASY-Blunt Simple Cloning Vector; the italicized part is the PAM sequence, and the underlined area is the targeting region.
  • the gRNA sequence is: GUGCUGCCGGUCUCUAAUCGGGGAUCGGAAUUGCAC CC CCCAGCGCUUCAGCGUUC (the underlined area is the targeting area); the following reaction system was used: the final concentration of Cas-sf19 was 100 nM, the final concentration of gRNA was 50 nM, and the final concentration of double-stranded target nucleic acid was 5 ng/ ⁇ L. Incubate for 1 h at 37°C. The experimental group added gRNA and target nucleic acid, and the control group did not add gRNA.

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Abstract

提供了一种可以在细胞内和细胞外表现出核酸酶的活性的新型Cas效应蛋白及其应用。

Description

CRISPR酶和系统以及应用 技术领域
本发明涉及基因编辑领域，特别是规律成簇的间隔短回文重复(CRISPR)技术领域。具体而言，本发明涉及CRISPR酶和系统以及应用，具体涉及一种新型的CRISPR酶(或者，称之为CRISPR蛋白、Cas效应蛋白、Cas酶或Cas蛋白)，包含此类蛋白的融合蛋白，以及编码它们的核酸分子。本发明还涉及用于核酸编辑(例如，基因或基因组编辑)的复合物和组合物，其包含本发明的Cas蛋白或融合蛋白，或编码它们的核酸分子。
背景技术
CRISPR/Cas技术是一种被广泛使用的基因编辑技术，它通过RNA引导对基因组上的靶序列进行特异性结合并切割DNA产生双链断裂，利用生物非同源末端连接或同源重组进行定点基因编辑。
CRISPR/Cas9系统是最常用的II型CRISPR系统，它识别3’-NGG的PAM基序，对靶标序列进行平末端切割。CRISPR/Cas Type V系统是一类新发现的CRISPR系统，它具有5’-TTN的基序，对靶标序列进行粘性末端切割，例如Cpf1,C2c1,CasX,CasY。然而目前存在的不同的CRISPR/Cas各有不同的优点和缺陷。例如Cas9,C2c1和CasX均需要两条RNA进行指导RNA，而Cpf1只需要一条指导RNA而且可以用来进行多重基因编辑。CasX具有980个氨基酸的大小，而常见的Cas9，C2c1，CasY和Cpf1通常大小在1300个氨基酸左右。此外，Cas9，Cpf1，CasX，CasY的PAM序列都比较复杂多样，而C2c1识别严谨的5’-TTN，因此它的靶标位点比其他系统容易被预测从而降低了潜在的脱靶效应。
总之，鉴于目前可获得的CRISPR/Cas系统都受限于一些缺陷，开发一种更稳健的、具有多方面良好性能的新型CRISPR/Cas系统对生物技术的发展具有重要意义。
发明内容
本申请的发明人经过大量实验和反复摸索，出人意料地发现了一种新型核酸内切酶(Cas酶)。基于这一发现，本发明人开发了新的CRISPR/Cas系统以及基 于该系统的基因编辑方法和核酸检测方法。
Cas效应蛋白
一方面，本发明提供了一种Cas蛋白，所述Cas蛋白是CRISPR/Cas系统中的效应蛋白，在本发明中，又将其称为Cas-sf19蛋白。
在一个实施方式中，所述Cas蛋白氨基酸序列与SEQ ID NO：1相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％的序列同一性，并且基本保留了SEQ ID NO:1的生物学功能。
在一个实施方式中，本发明所述的Cas蛋白与Cas-sf19蛋白来源于同一物种。
在一个实施方式中，所述Cas蛋白氨基酸序列与SEQ ID NO：1相比，具有一个或多个氨基酸的置换、缺失或添加的序列；所述一个或多个氨基酸包括1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加。
在一个实施方式中，本发明所述的Cas蛋白具有以下i-iii中的任意一个或任意几个功能结构域：
i、PVSVMGIDLGVNPAFAYAVCT；
ii、KSYIDYYKNLRLDTLKKLTCAIVRTARSHGVEIVALEDIKRVDYDDQVKRAKENSLLSLWAPGMILERIEQELANEGIRTWRIDPRHTSQTACITDEFGY；
iii、GELLRVNSDVNAAINIARRFLTR。
本领域技术人员清楚，可以改变蛋白质的结构而不对其活性和功能性产生不利影响，例如，可以在蛋白质氨基酸序列中引入一个或多个保守性氨基酸取代，而不会对蛋白质分子的活性和/或三维结构产生不利影响。本领域技术人员清楚保守性氨基酸取代的实例以及实施方式。具体的说，可以用与待取代位点属于相同组的另一氨基酸残基取代该氨基酸残基，即用非极性氨基酸残基取代另一非极性氨基酸残基，用极性不带电荷的氨基酸残基取代另一极性不带电荷的氨基酸残基，用碱性氨基酸残基取代另一碱性氨基酸残基，和用酸性氨基酸残基取代另一酸性氨基酸残基。这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的。只要取代不导致蛋白质生物活性的失活，则一种氨基酸被属于同组的其他氨基酸替换的保守取代落在本发明的范围内。因此，本发明的蛋白可以在氨基酸序列中包 含一个或多个保守性取代,这些保守性取代最好根据表1进行替换而产生。另外，本发明也涵盖还包含一个或多个其他非保守取代的蛋白，只要该非保守取代不显著影响本发明的蛋白质的所需功能和生物活性即可。
保守氨基酸置换可以在一个或多个预测的非必需氨基酸残基处进行。“非必需”氨基酸残基是可以发生改变(缺失、取代或置换)而不改变生物活性的氨基酸残基，而“必需”氨基酸残基是生物活性所需的。“保守氨基酸置换”是其中氨基酸残基被具有类似侧链的氨基酸残基替代的置换。氨基酸置换可以在Cas-sf19的非保守区域中进行。一般而言，此类置换不对保守的氨基酸残基，或者不对位于保守基序内的氨基酸残基进行，其中此类残基是蛋白质活性所需的。然而，本领域技术人员应当理解，功能变体可以具有较少的在保守区域中的保守或非保守改变。
表1
Figure PCTCN2022075030-appb-000001
本领域熟知，可以从蛋白质的N和/或C末端改变(置换、删除、截短或插入)一或多个氨基酸残基而仍保留其功能活性。因此，从Cas蛋白的N和/或C末端改 变了一或多个氨基酸残基、同时保留了其所需功能活性的蛋白，也在本发明的范围内。这些改变可以包括通过现代分子方法例如PCR而引入的改变，所述方法包括借助于在PCR扩增中使用的寡核苷酸之中包含氨基酸编码序列而改变或延长蛋白质编码序列的PCR扩增。
应认识到，蛋白质可以以各种方式进行改变，包括氨基酸置换、删除、截短和插入，用于此类操作的方法是本领域通常已知的。例如，可以通过对DNA的突变来制备Cas-sf19蛋白的氨基酸序列变体。还可以通过其他诱变形式和/或通过定向进化来完成，例如，使用已知的诱变、重组和/或改组(shuffling)方法，结合相关的筛选方法，来进行单个或多个氨基酸取代、缺失和/或插入。
领域技术人员能够理解，本发明Cas-sf19蛋白中的这些微小氨基酸变化可以出现(例如天然存在的突变)或者产生(例如使用r-DNA技术)而不损失蛋白质功能或活性。如果这些突变出现在蛋白的催化结构域、活性位点或其它功能结构域中，则多肽的性质可改变，但多肽可保持其活性。如果存在的突变不接近催化结构域、活性位点或其它功能结构域中，则可预期较小影响。
本领域技术人员可以根据本领域已知的方法，例如定位诱变或蛋白进化或生物信息系的分析，来鉴定Cas-sf19蛋白的必需氨基酸。蛋白的催化结构域、活性位点或其它功能结构域也能够通过结构的物理分析而确定，如通过以下这些技术：如核磁共振、晶体学、电子衍射或光亲和标记，结合推定的关键位点氨基酸的突变来确定。
在一个实施方式中，所述Cas蛋白含有SEQ ID NO：1所示的氨基酸序列。
在一个实施方式中，所述Cas蛋白为SEQ ID NO：1所示的氨基酸序列。
在一个实施方式中，所述Cas蛋白是与具有SEQ ID NO：1所示的序列的蛋白质相同生物学功能的衍生化蛋白。
所述生物学功能包括但不限于，与指导RNA结合的活性、核酸内切酶活性、在指导RNA引导下与靶序列特定位点结合并切割的活性，包括但不限于Cis切割活性和Trans切割活性。
本发明还提供了一种融合蛋白，所述融合蛋白包括如上所述的Cas蛋白和其他的修饰部分。
在一个实施方式中，所述修饰部分选自另外的蛋白或多肽、可检测的标记或 其任意组合。
在一个实施方式中，所述修饰部分选自表位标签、报告基因序列、核定位信号(NLS)序列、靶向部分、转录激活结构域(例如，VP64)、转录抑制结构域(例如，KRAB结构域或SID结构域)、核酸酶结构域(例如，Fok1)，以及具有选自下列的活性的结构域：核苷酸脱氨酶，甲基化酶活性,去甲基化酶,转录激活活性,转录抑制活性,转录释放因子活性,组蛋白修饰活性,核酸酶活性,单链RNA切割活性,双链RNA切割活性,单链DNA切割活性,双链DNA切割活性和核酸结合活性；以及其任意组合。所述NLS序列是本领域技术人员熟知的，其实例包括但不限于所述，SV40大T抗原，EGL-13，c-Myc以及TUS蛋白。
在一个实施方式中，所述NLS序列位于、靠近或接近本发明的Cas蛋白的末端(例如，N端、C端或两端)。
所述表位标签(epitope tag)是本领域技术人员熟知的，包括但不限于His、V5、FLAG、HA、Myc、VSV-G、Trx等，并且本领域技术人员可以选择其他合适的表位标签(例如，纯化、检测或示踪)。
所述报告基因序列是本领域技术人员熟知的，其实例包括但不限于GST、HRP、CAT、GFP、HcRed、DsRed、CFP、YFP、BFP等。
在一个实施方式中，本发明的融合蛋白包含能够与DNA分子或细胞内分子结合的结构域，例如麦芽糖结合蛋白(MBP)、Lex A的DNA结合结构域(DBD)、GAL4的DBD等。
在一个实施方式中，本发明的融合蛋白包含可检测的标记，例如荧光染料，例如FITC或DAPI。
在一个实施方式中，本发明的Cas蛋白任选地通过接头与所述修饰部分偶联、缀合或融合。
在一个实施方式中，所述修饰部分直接连接至本发明的Cas蛋白的N端或C端。
在一个实施方式中，所述修饰部分通过接头连接至本发明的Cas蛋白的N端或C端。这类接头是本领域熟知的，其实例包括但不限于包含一个或多个(例如，1个，2个，3个，4个或5个)氨基酸(如，Glu或Ser)或氨基酸衍生物(如，Ahx、β-Ala、GABA或Ava)的接头，或PEG等。
本发明的Cas蛋白、蛋白衍生物或融合蛋白不受其产生方式的限定，例如，其可以通过基因工程方法(重组技术)产生，也可以通过化学合成方法产生。
Cas蛋白的核酸
另一方面，本发明提供了一种分离的多核苷酸，其包含：
(a)编码本发明的Cas蛋白或融合蛋白的多核苷酸序列；
(b)序列如SEQ ID NO：2所示的多核苷酸；
(c)与SEQ ID NO：2所示的序列相比具有一个或多个碱基的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个碱基的置换、缺失或添加)的序列；
(d)核苷酸序列与SEQ ID NO：2所示序列的同源性≥80％(较佳地≥90％，更佳地≥95％，最佳地≥98％)，且编码SEQ ID NO:1所示多肽的多核苷酸；或者，核苷酸序列与SEQ ID NO:2所示序列的同源性≥80％(较佳地≥90％，更佳地≥95％，最佳地≥98％)，且编码SEQ ID NO:1所示多肽的多核苷酸；或者，核苷酸序列与SEQ ID NO:2所示序列的同源性≥80％(较佳地≥90％，更佳地≥95％，最佳地≥98％)，且编码SEQ ID NO:1所示多肽的多核苷酸；或者，
(e)与(a)-(d)任一所述的多核苷酸互补的多核苷酸。
在一个实施方式中，(a)-(e)任一项中所述的核苷酸序列经密码子优化用于在原核细胞中进行表达。在一个实施方式中，(a)-(e)任一项中所述的核苷酸序列经密码子优化用于在真核细胞中进行表达。
在一个实施方式中，所述的多核苷酸优选是单链的或双链的。
同向重复(Direct Repeat)序列
另一方面，本发明提供了一种与上述Cas蛋白形成复合物的工程化同向重复序列。
所述同向重复序列与能够和靶序列杂交的引导序列连接后构成指导RNA(guide RNA或gRNA)。
所述靶序列与gRNA的杂交，代表靶序列和gRNA的核酸序列至少70％，75％，80％，85％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％，或100％的同一性，从而可以杂交形成复合物；或者代表靶序列和gRNA的核酸序列至少有12 个，15个，16个，17个，18个，19个，20个，21个，22个，或更多个碱基可以互补配对，形成复合物。
在一些实施例中，该同向重复序列与SEQ ID NO：3具有至少90％的序列同一性。在一些实施例中，该同向重复序列与SEQ ID NO：3所示的序列相比具有一个或多个碱基的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个碱基的置换、缺失或添加)的序列。
在一些实施例中，同向重复序列如SEQ ID NO：3所示。
指导RNA(gRNA)
另一方面，本发明提供了一种gRNA，所述gRNA包括第一区段和第二区段；所述第一区段又称为“骨架区”、“蛋白质结合区段”、“蛋白质结合序列”、或者“同向重复(Direct Repeat)序列”；所述第二区段又称为“靶向核酸的靶向序列”或者“靶向核酸的靶向区段”，或者“靶向靶序列的引导序列”。
所述gRNA的第一区段能够与本发明的Cas蛋白相互作用，从而使Cas蛋白和gRNA形成复合物。
本发明靶向核酸的靶向序列或靶向核酸的靶向区段包含与靶核酸中的序列互补的核苷酸序列。换言之，本发明靶向核酸的靶向序列或靶向核酸的靶向区段经过杂交(即，碱基配对)以序列特异性方式与靶核酸相互作用。因此，靶向核酸的靶向序列或靶向核酸的靶向区段可改变，或可被修饰以杂交靶核酸内的任何希望的序列。所述核酸选自DNA或RNA。
靶向核酸的靶向序列或靶向核酸的靶向区段与靶核酸的靶序列之间的互补百分比可为至少60％(例如，至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少97％、至少98％、至少99％或100％)。
本发明gRNA的“骨架区”、“蛋白质结合区段”、“蛋白质结合序列”、或者“同向重复序列”可以与CRISPR蛋白(或者，Cas蛋白)相互作用。本发明gRNA经过靶向核酸的靶向序列的作用将其相互作用的Cas蛋白引导至靶核酸内的特异性核苷酸序列。
优选的，所述指导RNA从5’至3’方向包含第一区段和第二区段。
本发明中，所述第二区段还可以理解为与靶序列杂交的引导序列。
本发明的gRNA能够与所述Cas蛋白形成复合物。
本发明的Cas蛋白的gRNA包含与靶序列杂交的引导序列，其中，所述靶序列位于原型间隔区相邻基序(PAM)的3’端，所述PAM序列为5’-ATG-3’。
载体
本发明还提供了一种载体，其包含如上述的Cas蛋白、分离的核酸分子或多核苷酸；优选的，其还包括与之可操作连接的调控元件。
在一个实施方式中，所述的调控元件选自下组中的一种或多种：增强子、转座子、启动子、终止子、前导序列、多腺苷酸序列、标记基因。
在一个实施方式中，所述的载体包括克隆载体、表达载体、穿梭载体、整合载体。
在一些实施方案中，所述系统中包括的载体是病毒载体(例如逆转录病毒载体，慢病毒载体，腺病毒载体，腺相关载体和单纯疱疹载体)，还可以是质粒、病毒、粘粒、噬菌体等类型，它们是本领域技术人员所熟知的。
载体系统
本发明提供了一种工程化的非天然存在的载体系统，或者是CRISPR-Cas系统，该系统包括Cas蛋白或编码所述Cas蛋白的核酸序列以及编码一种或多种指导RNA的核酸。
在一种实施方式中，所述编码所述Cas蛋白的核酸序列和编码一种或多种指导RNA的核酸是人工合成的。
在一种实施方式中，所述编码所述Cas蛋白的核酸序列和编码一种或多种指导RNA的核酸并不共同天然存在。
该一种或多种指导RNA在细胞中靶向一个或多个靶序列。所述一个或多个靶序列与编码一种或多种基因产物的DNA分子的基因组座位杂交，并且引导该Cas蛋白到达所述一种或多种基因产物的DNA分子的基因组座位部位，Cas蛋白到达靶序列位置后对靶序列进行修饰、编辑或切割，由此该一种或多种基因产物的表达被改变或修饰。
本发明的细胞包括动物、植物或微生物中的一种或多种。
在一些实施例中，该Cas蛋白是密码子优化的，用于在细胞中进行表达。
在一些实施例中，该Cas蛋白指导切割在该靶序列位置处的一条或两条链。
本发明还提供了一种工程化的非天然存在的载体系统，该载体系统可以包括一种或多种载体，该一种或多种载体包括：
a)第一调控元件，该第一调控元件可操作地与gRNA连接，
b)第二调控元件，该第二调控元件可操作地与所述Cas蛋白连接；
其中组分(a)和(b)位于该系统的相同或不同载体上。
所述第一和第二调控元件包括启动子(例如，组成型启动子或诱导型启动子)、增强子(例如35S promoter或35S enhanced promoter)、内部核糖体进入位点(IRES)、和其他表达控制元件(例如转录终止信号，如多聚腺苷酸化信号和多聚U序列)。
在一些实施方案中，所述系统中的载体是病毒载体(例如逆转录病毒载体，慢病毒载体，腺病毒载体，腺相关载体和单纯疱疹载体)，还可以是质粒、病毒、粘粒、噬菌体等类型，它们是本领域技术人员所熟知的。
在一些实施例中，本文提供的系统处于递送系统中。在一些实施方案中，递送系统是纳米颗粒，脂质体，外体，微泡和基因枪。
在一个实施方式中，所述靶序列是来自原核细胞或真核细胞的DNA或RNA序列。在一个实施方式中，所述靶序列是非天然存在的DNA或RNA序列。
在一个实施方式中，当所述靶序列为DNA时，所述靶序列位于原间隔序列临近基序(PAM)的3’端，并且所述PAM具有5’-ATG-3’所示的序列。
在一个实施方式中，所述靶序列存在于细胞内。在一个实施方式中，所述靶序列存在于细胞核内或细胞质(例如，细胞器)内。在一个实施方式中，所述细胞是真核细胞。在其他实施方式中，所述细胞是原核细胞。
在一个实施方式中，所述Cas蛋白连接有一个或多个NLS序列。在一个实施方式中，所述融合蛋白包含一个或多个NLS序列。在一个实施方式中，所述NLS序列连接至所述蛋白的N端或C端。在一个实施方式中，所述NLS序列融合至所述蛋白的N端或C端。
另一方面，本发明涉及一种工程化的CRISPR系统，所述系统包含上述Cas蛋白以及一种或多种指导RNA，其中，所述指导RNA包括同向重复序列和能够与靶核酸杂交的间隔序列，所述Cas蛋白能够结合所述指导RNA并靶向与间隔序列互补的靶核酸序列。
蛋白-核酸复合物/组合物
另一方面，本发明提供了一种复合物或者组合物，其包含：
(i)蛋白组分，其选自：上述Cas蛋白、衍生化蛋白或融合蛋白，及其任意组合；和
(ii)核酸组分，其包含(a)能够与靶序列杂交的引导序列；以及(b)能够与本发明的Cas蛋白结合的同向重复序列。
所述蛋白组分与核酸组分相互结合形成复合物。
在一个实施方式中，所述核酸组分是CRISPR-Cas系统中的指导RNA。
在一个实施方式中，所述复合物或组合物是非天然存在的或经修饰的。在一个实施方式中，所述复合物或组合物中的至少一个组分是非天然存在的或经修饰的。在一个实施方式中，所述第一组分是非天然存在的或经修饰的；和/或，所述第二组分是非天然存在的或经修饰的。
活化的CRISPR复合物
另一方面，本发明还提供了一种活化的CRISPR复合物，所述活化的CRISPR复合物包含：(1)蛋白组分，其选自：本发明的Cas蛋白、衍生化蛋白或融合蛋白，及其任意组合；(2)gRNA，其包含(a)能够与靶序列杂交的引导序列；以及(b)能够与本发明的Cas蛋白结合的同向重复序列；以及(3)结合在gRNA上的靶序列。优选的，所述结合为通过gRNA上的靶向核酸的靶向序列与靶核酸进行的结合。
本文所用术语“活化的CRISPR复合物”，“活化复合物”或“三元复合物”是指CRISPR系统中Cas蛋白、gRNA与靶核酸结合或修饰后的复合物。
本发明的Cas蛋白和gRNA可以形成二元复合物，该二元复合物在与核酸底物结合时被活化，形成活化的CRISPR复合物该核酸底物与gRNA中的间隔序列(或者称之为，与靶核酸杂交的引导序列)互补。在一些实施方案中，gRNA的间隔序列与靶底物完全匹配。在其它实施方案中，gRNA的间隔序列与靶底物的部分(连续或不连续)匹配。
在优选的实施方式中，所述活化的CRISPR复合物可以表现出侧枝核酸酶切活性，所述侧枝核酸酶切活性是指活化的CRISPR复合物表现的对单链核酸的非特异 切割活性或乱切活性，在本领域又称之为trans切割活性。
递送及递送组合物
本发明的Cas蛋白、gRNA、融合蛋白、核酸分子、载体、系统、复合物和组合物，可以通过本领域已知的任何方法进行递送。此类方法包括但不限于，电穿孔、脂转染、核转染、显微注射、声孔效应、基因枪、磷酸钙介导的转染、阳离子转染、脂质体转染、树枝状转染、热激转染、核转染、磁转染、脂转染、穿刺转染、光学转染、试剂增强性核酸摄取、以及经由脂质体、免疫脂质体、病毒颗粒、人工病毒体等的递送。
因此，在另一个方面，本发明提供了一种递送组合物，其包含递送载体，以及选自下列的一种或任意几种：本发明的Cas蛋白、融合蛋白、核酸分子、载体、系统、复合物和组合物。
在一个实施方式中，所述递送载体是粒子。
在一个实施方式中，所述递送载体选自脂质颗粒、糖颗粒、金属颗粒、蛋白颗粒、脂质体、外泌体、微泡、基因枪或病毒载体(例如，复制缺陷型逆转录病毒、慢病毒、腺病毒或腺相关病毒)。
宿主细胞
本发明还涉及一种体外的、离体的或体内的细胞或细胞系或它们的子代，所述细胞或细胞系或它们的子代包含：本发明所述的Cas蛋白、融合蛋白、核酸分子、蛋白-核酸复合物、活化的CRISPR复合物、载体、本发明递送组合物。
在某些实施方案中，所述细胞是原核细胞。
在某些实施方案中，所述细胞是真核细胞。在某些实施方案中，所述细胞是哺乳动物细胞。在某些实施方案中，所述细胞是人类细胞。某些实施方案中，所述细胞是非人哺乳动物细胞，例如非人灵长类动物、牛、羊、猪、犬、猴、兔、啮齿类(如大鼠或小鼠)的细胞。在某些实施方案中，所述细胞是非哺乳动物真核细胞，例如家禽鸟类(如鸡)、鱼类或甲壳动物(如蛤蜊、虾)的细胞。在某些实施方案中，所述细胞是植物细胞，例如单子叶植物或双子叶植物具有的细胞或栽培植物或粮食作物如木薯、玉米、高粱、大豆、小麦、燕麦或水稻具有的细胞，例如藻类、树或生产植物、果实或蔬菜(例如，树类如柑橘树、坚果树；茄 属植物、棉花、烟草、番茄、葡萄、咖啡、可可等)。
在某些实施方案中，所述细胞是干细胞或干细胞系。
在某些情况下，本发明的宿主细胞包含基因或基因组的修饰，该修饰是在其野生型中不存在的修饰。
基因编辑方法和应用
本发明的Cas蛋白、核酸、上述组合物、上述CIRSPR/Cas系统、上述载体系统、上述递送组合物或上述活化的CRISPR复合物或者上述宿主细胞可用于以下任一或任意几个用途：靶向和/或编辑靶核酸；切割双链DNA、单链DNA或单链RNA；非特异性切割和/或降解侧枝核酸；非特异性切割单链核酸；核酸检测；检测目标样品中的核酸；特异性地编辑双链核酸；碱基编辑双链核酸；碱基编辑单链核酸。在其他的实施方式中，还可以用于制备用于上述任一或任意几个用途的试剂或试剂盒。
本发明还提供了上述Cas蛋白、核酸、上述组合物、上述CIRSPR/Cas系统、上述载体系统、上述递送组合物或上述活化的CRISPR复合物在基因编辑、基因靶向或基因切割中的应用；或者，在制备用于基因编辑、基因靶向或基因切割的试剂或试剂盒中的用途。
在一个实施方式中，所述基因编辑、基因靶向或基因切割为在细胞内和/或细胞外进行基因编辑、基因靶向或基因切割。
本发明还提供了一种编辑靶核酸、靶向靶核酸或切割靶核酸的方法，所述方法包括将靶核酸与上述Cas蛋白、核酸、上述组合物、上述CIRSPR/Cas系统、上述载体系统、上述递送组合物或上述活化的CRISPR复合物进行接触。在一个实施方式中，所述方法为在细胞内或细胞外编辑靶核酸、靶向靶核酸或切割靶核酸。
所述基因编辑或编辑靶核酸包括修饰基因、敲除基因、改变基因产物的表达、修复突变、和/或插入多核苷酸、基因突变。
所述编辑可以在原核细胞和/或真核细胞中进行编辑。
另一方面，本发明还提供了上述Cas蛋白、核酸、上述组合物、上述CIRSPR/Cas系统、上述载体系统、上述递送组合物或上述活化的CRISPR复合物在核酸检测中的应用，或在制备用于核酸检测的试剂或试剂盒中的用途。
另一方面，本发明还提供了一种切割单链核酸的方法，所述方法包括，使核 酸群体与上述Cas蛋白和gRNA接触，其中所述核酸群体包含靶核酸和多个非靶单链核酸，所述Cas蛋白切割所述多个非靶单链核酸。
所述gRNA能够结合所述Cas蛋白。
所述gRNA能够靶向所述靶核酸。
所述接触可以是在体外、离体或体内的细胞内部。
优选的，所述切割单链核酸为非特异性的切割。
另一方面，本发明还提供了上述Cas蛋白、核酸、上述组合物、上述CIRSPR/Cas系统、上述载体系统、上述递送组合物或上述活化的CRISPR复合物在非特异性的切割单链核酸中的应用，或在制备用于非特异性的切割单链核酸的试剂或试剂盒中的用途。
另一方面，本发明还提供了一种用于基因编辑、基因靶向或基因切割的试剂盒，所述试剂盒包括上述Cas蛋白、gRNA、核酸、上述组合物、上述CIRSPR/Cas系统、上述载体系统、上述递送组合物、上述活化的CRISPR复合物或上述宿主细胞。
另一方面，本发明还提供了一种用于检测样品中的靶核酸的试剂盒，所述试剂盒包含：(a)Cas蛋白，或编码所述Cas蛋白的核酸；(b)指导RNA，或编码所述指导RNA的核酸，或包含所述指导RNA的前体RNA，或编码所述前体RNA的核酸；和(c)为单链的且不与所述指导RNA杂交的单链核酸检测器。
本领域知晓，前体RNA可被切割或加工成为上述成熟的指导RNA。
另一方面，发明提供了上述Cas蛋白、核酸、上述组合物、上述CIRSPR/Cas系统、上述载体系统、上述递送组合物、上述活化的CRISPR复合物或上述宿主细胞在制备制剂或试剂盒中的用途，所述制剂或试剂盒用于：
(i)基因或基因组编辑；
(ii)靶核酸检测和/或诊断；
(iii)编辑靶基因座中的靶序列来修饰生物或非人类生物；
(iv)疾病的治疗；
(iv)靶向靶基因。
优选的，上述基因或基因组编辑为在细胞内或细胞外进行基因或基因组编辑。
优选的，所述靶核酸检测和/或诊断为在体外进行靶核酸检测和/或诊断。
优选的，所述疾病的治疗为治疗由靶基因座中的靶序列的缺陷引起的病症。
另一个方面，本发明提供了一种检测样品中靶核酸的方法，所述方法包括将样品与所述Cas蛋白、gRNA(指导RNA)和单链核酸检测器接触，所述gRNA包括与所述Cas蛋白结合的区域和与靶核酸杂交的指导序列；检测由所述Cas蛋白切割单链核酸检测器产生的可检测信号，从而检测靶核酸；所述单链核酸检测器不与所述gRNA杂交。
特异性修饰靶核酸的方法
另一方面，本发明还提供了一种特异性修饰靶核酸的方法，方法包括：使靶核酸与上述Cas蛋白、核酸、上述组合物、上述CIRSPR/Cas系统、上述载体系统、上述递送组合物或上述活化的CRISPR复合物接触。
该特异性修饰可以发生在体内或者体外。
该特异性修饰可以发生在细胞内或者细胞外。
在一些情况下，细胞选自原核细胞或真核细胞，例如，动物细胞、植物细胞或微生物细胞。
在一个实施方式中，所述修饰是指所述靶序列的断裂，如，DNA的单链/双链断裂，或者RNA的单链断裂。
在一些情况下，所述方法还包括使靶核酸与供体多核苷酸接触，其中将供体多核苷酸、供体多核苷酸的部分、供体多核苷酸的拷贝或供体多核苷酸的拷贝的部分整合到靶核酸中。
在一个实施方式中，所述修饰还包括将编辑模板(例如外源核酸)插入所述断裂中。
在一个实施方式中，所述方法还包括：将编辑模板与所述靶核酸接触，或者递送至包含所述靶核酸的细胞中。在此实施方式中，所述方法通过与外源模板多核苷酸同源重组修复所述断裂的靶基因；在一些实施方式中，所述修复导致一种突变，包括所述靶基因的一个或多个核苷酸的插入、缺失、或取代，在其他的实施方式中，所述突变导致在从包含该靶序列的基因表达的蛋白质中的一个或多个氨基酸改变。
检测(非特异切割)
另一方面，本发明提供了一种检测样品中靶核酸的方法，所述方法包括将样品与上述Cas蛋白、核酸、上述组合物、上述CIRSPR/Cas系统、上述载体系统、上述递送组合物或上述活化的CRISPR复合物和单链核酸检测器接触；检测由所述Cas蛋白切割单链核酸检测器产生的可检测信号，从而检测靶核酸。
本发明中，所述靶核酸包括核糖核苷酸或脱氧核糖核苷酸；包括单链核酸、双链核酸，例如单链DNA、双链DNA、单链RNA、双链RNA。
在一个实施方式中，所述靶核酸来源于病毒、细菌、微生物、土壤、水源、人体、动物、植物等样品。优选的，所述靶核酸为PCR、NASBA、RPA、SDA、LAMP、HAD、NEAR、MDA、RCA、LCR、RAM等方法富集或扩增的产物。
在一个实施方式中，所述靶核酸为病毒核酸、细菌核酸、与疾病相关的特异核酸，如特定的突变位点或SNP位点或与对照有差异的核酸；优选地，所述病毒为植物病毒或动物病毒，例如，乳头瘤病毒，肝DNA病毒，疱疹病毒，腺病毒，痘病毒，细小病毒，冠状病毒；优选地，所述病毒为冠状病毒，优选地，SARS、SARS-CoV2(COVID-19)、HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、Mers-Cov。
本发明中，所述gRNA与靶核酸上的靶序列至少有50％的匹配度，优选至少60％，优选至少70％，优选至少80％，优选至少90％。
在一个实施方式中，当所述的靶序列含有一个或多个特征位点(如特定的突变位点或SNP)时，所述的特征位点与gRNA完全匹配。
在一个实施方式中，所述检测方法中可以包含一种或多种导向序列互不相同的gRNA，其靶向不同的靶序列。
本发明中，所述单链核酸检测器包括但不限于单链DNA、单链RNA、DNA-RNA杂交体、核酸类似物、碱基修饰物、以及含有无碱基间隔物的单链核酸检测器等；“核酸类似物”包括但不限于：锁核酸、桥核酸、吗啉核酸、乙二醇核酸、己糖醇核酸、苏糖核酸、阿拉伯糖核酸、2’氧甲基RNA、2’甲氧基乙酰基RNA、2’氟RNA、2’氨基RNA、4’硫RNA及其组合，包括任选的核糖核苷酸或脱氧核糖核苷酸残基。
本发明中，所述可检测信号通过以下方式实现：基于视觉的检测，基于传感器的检测，颜色检测，基于荧光信号的检测，基于金纳米颗粒的检测，荧光偏振， 胶体相变/分散，电化学检测和基于半导体的检测。
本发明中，优选的，所述单链核酸检测器的两端分别设置荧光基团和淬灭基团，当所述单链核酸检测器被切割后，可以表现出可检测的荧光信号。所述荧光基团选自FAM、FITC、VIC、JOE、TET、CY3、CY5、ROX、Texas Red或LC RED460中的一种或任意几种；所述淬灭基团选自BHQ1、BHQ2、BHQ3、Dabcy1或Tamra中的一种或任意几种。
在其他的实施方式中，所述单链核酸检测器的5’端和3’端分别设置不同的标记分子，通过胶体金检测的方式，检测所述单链核酸检测器被Cas蛋白切割前和被Cas蛋白切割后的胶体金测试结果；所述单链核酸检测器被Cas蛋白切割前和被Cas蛋白切割后在胶体金的检测线和质控线上将表现出不同的显色结果。
在一些实施方案中，检测靶核酸的方法还可以包括将可检测信号的电平与参考信号电平进行比较，以及基于可检测信号的电平确定样品中靶核酸的量。
在一些实施方案中，检测靶核酸的方法还可以包括在不同的通道上使用RNA报告核酸和DNA报告核酸(例如，荧光颜色)，并通过测量RNA和DNA报告分子的信号电平，以及通过测量RNA和DNA报告分子中靶核酸的量来确定可检测信号的电平，基于组合(例如，使用最小或乘积)可检测信号的电平来采样。
在一个实施方式中，所述靶基因存在于细胞内。
在一个实施方式中，所述细胞是原核细胞。
在一个实施方式中，所述细胞是真核细胞。
在一个实施方式中，所述细胞是动物细胞。
在一个实施方式中，所述细胞是人类细胞。
在一个实施方式中，所述细胞是植物细胞，例如栽培植物(如木薯、玉米、高粱、小麦或水稻)、藻类、树或蔬菜具有的细胞。
在一个实施方式中，所述靶基因存在于体外的核酸分子(例如，质粒)中。
在一个实施方式中，所述靶基因存在于质粒中。
术语定义
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的分子遗传学、核酸化学、化学、分子生物学、生物化学、细胞培养、微生物学、细胞生物学、基因组学和重组DNA 等操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
Cas蛋白
在本发明中，Cas蛋白、Cas酶、Cas效应蛋白可以互换使用；本发明人首次发现并鉴定了一种Cas效应蛋白，其具有选自下列的氨基酸序列：
(i)SEQ ID NO：1所示的序列；
(ii)与SEQ ID NO：1所示的序列相比具有一个或多个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加)的序列；或
(iii)与SEQ ID NO：1所示的序列具有至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％的序列同一性的序列。
本文中的核酸切割或切割核酸包括:由本文所述Cas酶产生的靶核酸中的DNA或RNA断裂(Cis切割)、DNA或RNA在侧枝核酸底物(单链核酸底物)中的断裂(即非特异性或非靶向性，Trans切割)。在一些实施方式中，所述切割是双链DNA断裂。在一些实施方案中，切割是单链DNA断裂或单链RNA断裂。
CRISPR系统
如本文中所使用的，术语“规律成簇的间隔短回文重复(CRISPR)-CRISPR-相关(Cas)(CRISPR-Cas)系统”或“CRISPR系统”可互换地使用并且具有本领域技术人员通常理解的含义，其通常包含与CRISPR相关(“Cas”)基因的表达有关的转录产物或其他元件，或者能够指导所述Cas基因活性的转录产物或其他元件。
CRISPR/Cas复合物
如本文中所使用的，术语“CRISPR/Cas复合物”是指，指导RNA(guide RNA)或成熟crRNA与Cas蛋白结合所形成的复合体，其包含杂交到靶序列的引导序列上并且与Cas蛋白结合的同向重复序列，该复合体能够识别并切割能与该指导RNA或成熟crRNA杂交的多核苷酸。
指导RNA(guide RNA，gRNA)
如本文中所使用的，术语“指导RNA(guide RNA，gRNA)”、“成熟crRNA”、 “指导序列”可互换地使用并且具有本领域技术人员通常理解的含义。一般而言，指导RNA可以包含同向重复序列(direct repeat)和引导序列，或者基本上由或由同向重复序列和引导序列组成。
在某些情况下，指导序列是与靶序列具有足够互补性从而与所述靶序列杂交并引导CRISPR/Cas复合物与所述靶序列的特异性结合的任何多核苷酸序列。在一个实施方式中，当最佳比对时，指导序列与其相应靶序列之间的互补程度为至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、或至少99％。确定最佳比对在本领域的普通技术人员的能力范围内。例如，存在公开和可商购的比对算法和程序，诸如但不限于ClustalW、matlab中的史密斯-沃特曼算法(Smith-Waterman)、Bowtie、Geneious、Biopython以及SeqMan。
靶序列
“靶序列”是指被gRNA中的引导序列所靶向的多核苷酸，例如与该引导序列具有互补性的序列，其中靶序列与引导序列之间的杂交将促进CRISPR/Cas复合物(包括Cas蛋白和gRNA)的形成。完全互补性不是必需的，只要存在足够互补性以引起杂交并且促进一种CRISPR/Cas复合物的形成即可。
靶序列可以包含任何多核苷酸，如DNA或RNA。在某些情况下，所述靶序列位于细胞内或细胞外。在某些情况下，所述靶序列位于细胞的细胞核或细胞质中。在某些情况下，该靶序列可位于真核细胞的一个细胞器例如线粒体或叶绿体内。可被用于重组到包含该靶序列的靶基因座中的序列或模板被称为“编辑模板”或“编辑多核苷酸”或“编辑序列”。在一个实施方式中，所述编辑模板为外源核酸。在一个实施方式中，该重组是同源重组。
在本发明中，“靶序列”或“靶多核苷酸”或“靶核酸”可以是对细胞(例如，真核细胞)而言任何内源或外源的多核苷酸。例如，该靶多核苷酸可以是一种存在于真核细胞的细胞核中的多核苷酸。该靶多核苷酸可以是一个编码基因产物(例如，蛋白质)的序列或一个非编码序列(例如，调节多核苷酸或无用DNA)。在某些情况下，该靶序列应该与原间隔序列临近基序(PAM)相关。
单链核酸检测器
本发明所述的单链核酸检测器是指含有2-200个核苷酸的序列，优选，具有2-150个核苷酸，优选，3-100个核苷酸，优选，3-30个核苷酸，优选，4-20个 核苷酸，更优选，5-15个核苷酸。优选为单链DNA分子、单链RNA分子或单链DNA-RNA杂交体。
所述的单链核酸检测器两端包括不同的报告基团或标记分子，当其处于初始状态(即未被切割状态时)不呈现报告信号，当该单链核酸检测器被切割后，呈现出可检测的信号，即切割后与切割前表现出可检测的区别。
在一个实施方式中，所述的报告基团或标记分子包括荧光基团和淬灭基团，所述荧光基团选自FAM、FITC、VIC、JOE、TET、CY3、CY5、ROX、Texas Red或LC RED460中的一种或任意几种；所述淬灭基团选自BHQ1、BHQ2、BHQ3、Dabcy1或Tamra中的一种或任意几种。
在一个实施方式中，所述的单链核酸检测器具有连接至5’端第一分子(如FAM或FITC)和连接至3’端的第二分子(如生物素)。所述的含有单链核酸检测器的反应体系与流动条配合用以检测靶核酸(优选，胶体金检测方式)。所述的流动条被设计为具有两条捕获线，在样品接触端(胶体金)设有结合第一分子的抗体(即第一分子抗体)，在第一线(control line)处含有结合第一分子抗体的抗体，在第二线(test line)处含有与第二分子结合的第二分子的抗体(即第二分子抗体，如亲和素)。当反应沿着条带流动时，第一分子抗体与第一分子结合携带切割或未切割的寡核苷酸至捕获线，切割的报告子将在第一个捕获线处结合第一分子抗体的抗体，而未切割的报告子将在第二捕获线处结合第二分子抗体。报告基团在各条线的结合将导致强读出/信号(例如颜色)。随着更多的报告子被切割，更多的信号将在第一捕获线处累积，并且在第二线处将出现更少的信号。在某些方面，本发明涉及如本文所述的流动条用于检测核酸的用途。在某些方面，本发明涉及用本文定义的流动条检测核酸的方法，例如(侧)流测试或(侧)流免疫色谱测定。在某些方面，所述单链核酸检测器中的分子可相互替换，或改变分子的位置，只要其报告原理与本发明相同或相近，所改进的方式也均包含在本发明中。
本发明所述的检测方法，可用于待检测靶核酸的定量检测。所述的定量检测指标可以根据报告基团的信号强弱进行定量，如根据荧光基团的发光强度，或根据显色条带的宽度等。
野生型
如本文中所使用的，术语“野生型”具有本领域技术人员通常理解的含义，其表示生物、菌株、基因的典型形式或者当它在自然界存在时区别于突变体或变体形式的特征，其可从自然中的来源分离并且没有被人为有意地修饰。
衍生化
如本文中所使用的，术语“衍生化”是指，对氨基酸、多肽或蛋白的化学修饰，其中一个或多个取代基已与所述氨基酸、多肽或蛋白共价连接。取代基也可称为侧链。
衍生化的蛋白是该蛋白的衍生物，通常，蛋白的衍生化不会不利影响该蛋白的期望活性(例如，与指导RNA结合的活性、核酸内切酶活性、在指导RNA引导下与靶序列特定位点结合并切割的活性)，也就是说蛋白的衍生物与蛋白有相同的活性。
衍生化蛋白
又称“蛋白衍生物”，是指蛋白的经修饰形式，例如其中所述蛋白的一个或多个氨基酸可以被缺失、插入、修饰和/或取代。
非天然存在的
如本文中所使用的，术语“非天然存在的”或“工程化的”可互换地使用并且表示人工的参与。当这些术语用于描述核酸分子或多肽时，其表示该核酸分子或多肽至少基本上从它们在自然界中或如发现于自然界中的与其结合的至少另一种组分游离出来。
直系同源物(orthologue,ortholog)
如本文中所使用的，术语“直系同源物(orthologue,ortholog)”具有本领域技术人员通常理解的含义。作为进一步指导，如本文中所述的蛋白质的“直系同源物”是指属于不同物种的蛋白质，该蛋白质执行与作为其直系同源物的蛋白相同或相似的功能。
同一性
如本文中所使用的，术语“同一性”用于指两个多肽之间或两个核酸之间序列的匹配情况。当两个进行比较的序列中的某个位置都被相同的碱基或氨基酸单体亚单元占据时(例如，两个DNA分子的每一个中的某个位置都被腺嘌呤占据，或两个多肽的每一个中的某个位置都被赖氨酸占据)，那么各分子在该位置上是同一 的。两个序列之间的“百分数同一性”是由这两个序列共有的匹配位置数目除以进行比较的位置数目×100的函数。例如，如果两个序列的10个位置中有6个匹配，那么这两个序列具有60％的同一性。例如，DNA序列CTGACT和CAGGTT共有50％的同一性(总共6个位置中有3个位置匹配)。通常，在将两个序列比对以产生最大同一性时进行比较。这样的比对可通过使用，例如，可通过计算机程序例如Align程序(DNAstar,Inc.)方便地进行的Needleman等人(1970)J.Mol.Biol.48：443-453的方法来实现。还可使用已整合入ALIGN程序(版本2.0)的E.Meyers和W.Miller(Comput.Appl Biosci.，4:11-17(1988))的算法，使用PAM120权重残基表(weight residue table)、12的缺口长度罚分和4的缺口罚分来测定两个氨基酸序列之间的百分数同一性。此外，可使用已整合入GCG软件包(可在www.gcg.com上获得)的GAP程序中的Needleman和Wunsch(J MoI Biol.48:444-453(1970))算法，使用Blossum 62矩阵或PAM250矩阵以及16、14、12、10、8、6或4的缺口权重(gap weight)和1、2、3、4、5或6的长度权重来测定两个氨基酸序列之间的百分数同一性。
载体
术语“载体”是指一种核酸分子，它能够运送与其连接的另一种核酸分子。载体包括但不限于，单链、双链、或部分双链的核酸分子；包括一个或多个自由端、无自由端(例如环状的)的核酸分子；包括DNA、RNA、或两者的核酸分子；以及本领域已知的其他多种多样的多核苷酸。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。一种载体可以被引入到宿主细胞中而由此产生转录物、蛋白质、或肽，包括由如本文所述的蛋白、融合蛋白、分离的核酸分子等(例如，CRISPR转录物，如核酸转录物、蛋白质、或酶)。一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。
一种类型的载体是“质粒”，其是指其中可以例如通过标准分子克隆技术插入另外的DNA片段的环状双链DNA环。
另一种类型的载体是病毒载体，其中病毒衍生的DNA或RNA序列存在于用于包装病毒(例如，逆转录病毒、复制缺陷型逆转录病毒、腺病毒、复制缺陷型腺病 毒、以及腺相关病毒)的载体中。病毒载体还包含由用于转染到一种宿主细胞中的病毒携带的多核苷酸。某些载体(例如，具有细菌复制起点的细菌载体和附加型哺乳动物载体)能够在它们被导入的宿主细胞中自主复制。
其他载体(例如，非附加型哺乳动物载体)在引入宿主细胞后整合到该宿主细胞的基因组中，并且由此与该宿主基因组一起复制。而且，某些载体能够指导它们可操作连接的基因的表达。这样的载体在此被称为“表达载体”。
宿主细胞
如本文中所使用的，术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌或枯草菌等的原核细胞，如微生物细胞、真菌细胞、动物细胞和植物细胞的真核细胞。
本领域技术人员将理解，表达载体的设计可取决于诸如待转化的宿主细胞的选择、所希望的表达水平等因素。
调控元件
如本文中所使用的，术语“调控元件”旨在包括启动子、增强子、内部核糖体进入位点(IRES)、和其他表达控制元件(例如转录终止信号，如多聚腺苷酸化信号和多聚U序列)，其详细描述可参考戈德尔(Goeddel)，《基因表达技术：酶学方法》(GENE EXPRESSION TECHNOLOGY:METHODS IN ENZYMOLOGY)185，学术出版社(Academic Press)，圣地亚哥(San Diego)，加利福尼亚州(1990)。在某些情况下，调控元件包括指导一个核苷酸序列在许多类型的宿主细胞中的组成型表达的那些序列以及指导该核苷酸序列只在某些宿主细胞中表达的那些序列(例如，组织特异型调节序列)。组织特异型启动子可主要指导在感兴趣的期望组织中的表达，所述组织例如肌肉、神经元、骨、皮肤、血液、特定的器官(例如肝脏、胰腺)、或特殊的细胞类型(例如淋巴细胞)。在某些情况下，调控元件还可以时序依赖性方式(如以细胞周期依赖性或发育阶段依赖性方式)指导表达，该方式可以是或者可以不是组织或细胞类型特异性的。在某些情况下，术语“调控元件”涵盖的是增强子元件，如WPRE；CMV增强子；在HTLV-I的LTR中的R-U5’片段((Mol.Cell.Biol.，第8(1)卷，第466-472页，1988)；SV40增强子；以及在兔β-珠蛋白的外显子2与3之间的内含子序列(Proc.Natl.Acad.Sci.USA.，第78(3)卷，第1527-31页，1981)。
启动子
如本文中所使用的，术语“启动子”具有本领域技术人员公知的含义，其是指一段位于基因的上游能启动下游基因表达的非编码核苷酸序列。组成型(constitutive)启动子是这样的核苷酸序列：当其与编码或者限定基因产物的多核苷酸可操作地相连时，在细胞的大多数或者所有生理条件下，其导致细胞中基因产物的产生。诱导型启动子是这样的核苷酸序列，当可操作地与编码或者限定基因产物的多核苷酸相连时，基本上只有当对应于所述启动子的诱导物在细胞中存在时，其导致所述基因产物在细胞内产生。组织特异性启动子是这样的核苷酸序列：当可操作地与编码或者限定基因产物的多核苷酸相连时，基本上只有当细胞是该启动子对应的组织类型的细胞时，其才导致在细胞中产生基因产物。
NLS
“核定位信号”或“核定位序列”(NLS)是对蛋白质“加标签”以通过核转运导入细胞核的氨基酸序列，即，具有NLS的蛋白质被转运至细胞核。典型地，NLS包含暴露在蛋白质表面的带正电荷的Lys或Arg残基。示例性核定位序列包括但不限于来自以下的NLS：SV40大T抗原，EGL-13，c-Myc以及TUS蛋白。在一些实施例中，该NLS包含PKKKRKV序列。在一些实施例中，该NLS包含AVKRPAATKKAGQAKKKKLD序列。在一些实施例中，该NLS包含PAAKRVKLD序列。在一些实施例中，该NLS包含MSRRRKANPTKLSENAKKLAKEVEN序列。在一些实施例中，该NLS包含KLKIKRPVK序列。其他核定位序列包括但不限于hnRNP A1的酸性M9结构域、酵母转录抑制子Matα2中的序列KIPIK和PY-NLS。
可操作地连接
如本文中所使用的，术语“可操作地连接”旨在表示感兴趣的核苷酸序列以一种允许该核苷酸序列的表达的方式被连接至该一种或多种调控元件(例如，处于一种体外转录/翻译系统中或当该载体被引入到宿主细胞中时，处于该宿主细胞中)。
互补性
如本文中所使用的，术语“互补性”是指核酸与另一个核酸序列借助于传统的沃森-克里克或其他非传统类型形成一个或多个氢键的能力。互补百分比表示一个核酸分子中可与一个第二核酸序列形成氢键(例如，沃森-克里克碱基配对)的残 基的百分比(例如，10个之中有5、6、7、8、9、10个即为50％、60％、70％、80％、90％、和100％互补)。“完全互补”表示一个核酸序列的所有连续残基与一个第二核酸序列中的相同数目的连续残基形成氢键。如本文使用的“基本上互补”是指在一个具有8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、30、35、40、45、50个或更多个核苷酸的区域上至少为60％、65％、70％、75％、80％、85％、90％、95％、97％、98％、99％、或100％的互补程度，或者是指在严格条件下杂交的两个核酸。
严格条件
如本文中所使用的，对于杂交的“严格条件”是指与靶序列具有互补性的一个核酸主要地与该靶序列杂交并且基本上不杂交到非靶序列上的条件。严格条件通常是序列依赖性的，并且取决于许多因素而变化。一般而言，该序列越长，则该序列特异性地杂交到其靶序列上的温度就越高。
杂交
术语“杂交”或“互补的”或“基本上互补的”是指核酸(例如RNA、DNA)包含使其能够非共价结合的核苷酸序列，即以序列特异性，反平行的方式(即核酸特异性结合互补核酸)与另一核酸形成碱基对和/或G/U碱基对，“退火”或“杂交”。
杂交需要两个核酸含有互补序列，尽管碱基之间可能存在错配。两个核酸之间杂交的合适条件取决于核酸的长度和互补程度，这是本领域公知的变量。典型地，可杂交核酸的长度为8个核苷酸或更多(例如，10个核苷酸或更多，12个核苷酸或更多，15个核苷酸或更多，20个核苷酸或更多，22个核苷酸或更多，25个核苷酸或更多，或30个核苷酸或更多)。
应当理解，多核苷酸的序列不需要与其靶核酸的序列100％互补以特异性杂交。多核苷酸可包含60％或更高，65％或更高，70％或更高，75％或更高，80％或更高，85％或更高，90％或更高，95％或更高，98％或更高，99％或更高，99.5％或更高，或与其杂交的靶核酸序列中的靶区域的序列互补性为100％。
靶序列与gRNA的杂交代表靶序列和gRNA的核酸序列至少60％、70％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的可以杂交，形成复合物；或者代表靶序列和gRNA的核酸序列至少有12个、15个、 16个、17个、18个、19个、20个、21个、22个或更多个碱基可以互补配对，杂交形成复合物。
表达
如本文中所使用的，术语“表达”是指，藉此从DNA模板转录成多核苷酸(如转录成mRNA或其他RNA转录物)的过程和/或转录的mRNA随后藉此翻译成肽、多肽或蛋白质的过程。转录物和编码的多肽可以总称为“基因产物”。如果多核苷酸来源于基因组DNA，表达可以包括真核细胞中mRNA的剪接。
接头
如本文中所使用的，术语“接头”是指，由多个氨基酸残基通过肽键连接形成的线性多肽。本发明的接头可以为人工合成的氨基酸序列，或天然存在的多肽序列，例如具有铰链区功能的多肽。此类接头多肽是本领域众所周知的(参见例如，Holliger,P.等人(1993)Proc.Natl.Acad.Sci.USA 90:6444-6448；Poljak,R.J.等人(1994)Structure 2:1121-1123)。
治疗
如本文中所使用的，术语“治疗”是指，治疗或治愈病症，延缓病症的症状的发作，和/或延缓病症的发展。
受试者
如本文中所使用的，术语“受试者”包括但不限于各种动物、植物和微生物。
动物
例如哺乳动物，例如牛科动物、马科动物、羊科动物、猪科动物、犬科动物、猫科动物、兔科动物、啮齿类动物(例如，小鼠或大鼠)、非人灵长类动物(例如，猕猴或食蟹猴)或人。在某些实施方式中，所述受试者(例如人)患有病症(例如，疾病相关基因缺陷所导致的病症)。
植物
术语“植物”应理解为能够进行光合作用的任何分化的多细胞生物，在包括处于任何成熟或发育阶段的作物植物，特别是单子叶或双子叶植物，蔬菜作物，包括洋蓟、球茎甘蓝、芝麻菜、韭葱、芦笋、莴苣(例如，结球莴苣、叶莴苣、长叶莴苣)、小白菜(bok choy)、黄肉芋、瓜类(例如，甜瓜、西瓜、克伦肖瓜(crenshaw)、白兰瓜、罗马甜瓜)、油菜作物(例如，球芽甘蓝、卷心菜、花椰菜、 西兰花、羽衣甘蓝、无头甘蓝、大白菜、小白菜)、刺菜蓟、胡萝卜、洋白菜(napa)、秋葵、洋葱、芹菜、欧芹、鹰嘴豆、欧洲防风草、菊苣、胡椒、马铃薯、葫芦(例如，西葫芦、黄瓜、小西葫芦、倭瓜、南瓜)、萝卜、干球洋葱、芜菁甘蓝、紫茄子(也称为茄子)、婆罗门参、苣菜、青葱、苦苣、大蒜、菠菜、绿洋葱、倭瓜、绿叶菜类(greens)、甜菜(糖甜菜和饲料甜菜)、甘薯、唐莴苣、山葵、西红柿、芜菁、以及香辛料；水果和/或蔓生作物，如苹果、杏、樱桃、油桃、桃、梨、李子、西梅、樱桃、榅桲、杏仁、栗子、榛子、山核桃、开心果、胡桃、柑橘、蓝莓、博伊增莓(boysenberry)、小红莓、穗醋栗、罗甘莓、树莓、草莓、黑莓、葡萄、鳄梨、香蕉、猕猴桃、柿子、石榴、菠萝、热带水果、梨果、瓜、芒果、木瓜、以及荔枝；大田作物，如三叶草、苜蓿、月见草、白芒花、玉米/玉蜀黍(饲料玉米、甜玉米、爆米花)、啤酒花、荷荷芭、花生、稻、红花、小粒谷类作物(大麦、燕麦、黑麦、小麦等)、高粱、烟草、木棉、豆科植物(豆类、小扁豆、豌豆、大豆)、含油植物(油菜、芥菜、罂粟、橄榄、向日葵、椰子、蓖麻油植物、可可豆、落花生)、拟南芥属、纤维植物(棉花、亚麻、大麻、黄麻)、樟科(肉桂、莰酮)、或一种植物如咖啡、甘蔗、茶、以及天然橡胶植物；和/或花坛植物，如开花植物、仙人掌、肉质植物和/或观赏植物，以及树如森林(阔叶树和常绿树，如针叶树)、果树、观赏树、以及结坚果的树(nut-bearing tree)、以及灌木和其他苗木。
发明的有益效果
本发明发现了一种新型的Cas酶，Blast结果显示，本申请的Cas酶与已经报道的Cas酶的一致性较低，其可以在体内和体外表现出核酸酶的活性，具有广泛的应用前景。
下面将结合附图和实施例对本发明的实施方案进行详细描述，但是本领域技术人员将理解，下列附图和实施例仅用于说明本发明，而不是对本发明的范围的限定。根据附图和优选实施方案的下列详细描述，本发明的各种目的和有利方面对于本领域技术人员来说将变得显然。
附图说明
图1.对Cas-sf19活性结构域的预测。
图2.Cas-sf19用于核酸检测的荧光结果图。
图3.Cas-sf19的PAM鉴定结果。
图4.Cas-sf19用于双链靶核酸检测的荧光结果图。
图5.Cas-sf19用于双链核酸的切割结果图。
序列信息
SEQ ID NO: 描述
1 Cas-sf19的氨基酸序列
2 Cas-sf19的核酸序列
3 Cas-sf19的gRNA的DR区
具体实施方式
以下实施例仅用于描述本发明，而非限定本发明。除非特别指明，否则基本上按照本领域内熟知的以及在各种参考文献中描述的常规方法进行实施例中描述的实验和方法。例如，本发明中所使用的免疫学、生物化学、化学、分子生物学、微生物学、细胞生物学、基因组学和重组DNA等常规技术，可参见萨姆布鲁克(Sambrook)、弗里奇(Fritsch)和马尼亚蒂斯(Maniatis)，《分子克隆：实验室手册》(MOLECULAR CLONING:A LABORATORY MANUAL)，第2次编辑(1989)；《当代分子生物学实验手册》(CURRENT PROTOCOLS IN MOLECULAR BIOLOGY)(F.M.奥苏贝尔(F.M.Ausubel)等人编辑，(1987))；《酶学方法》(METHODS IN ENZYMOLOGY)系列(学术出版公司)：《PCR 2：实用方法》(PCR 2:A PRACTICAL APPROACH)(M.J.麦克弗森(M.J.MacPherson)、B.D.黑姆斯(B.D.Hames)和G.R.泰勒(G.R.Taylor)编辑(1995))、哈洛(Harlow)和拉内(Lane)编辑(1988)《抗体：实验室手册》(ANTIBODIES,A LABORATORY MANUAL)，以及《动物细胞培养》(ANIMAL CELL CULTURE)(R.I.弗雷谢尼(R.I.Freshney)编辑(1987))。
另外，实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。本领域技术人员知晓，实施例以举例方式描述本发明，且不意欲限制本发明所要求保护 的范围。本文中提及的全部公开案和其他参考资料以其全文通过引用合并入本文。
实施例1.Cas蛋白的获得
发明人对未培养物的宏基因组进行分析，通过对去冗余、蛋白质聚类分析，鉴定得到了一种新的Cas酶，其氨基酸序列如SEQ ID NO：1所示，其核酸序列如SEQ ID NO：2所示。Blast结果显示，该Cas蛋白与已报道的Cas蛋白的序列一致性较低，本发明中将其命名为Cas-sf19。
分析发现，该Cas-sf19蛋白所对应的gRNA的同向重复序列为GUGCUGCCGGUCUCUAAUCGGGGAUCGGAAUUGCAC。
Cas-sf19蛋白包含911个氨基酸，与Pfam数据库中的结构域进行对比分析，分析结果如图1所示；预测到三个功能结构域，结构域为RuvC_1(PF18516)RuvC nuclease domain，该结构域具有内切核糖核酸酶活性，Cas-sf19蛋白序列中，3个RuvC nuclease domain的信息如下：
(1)472-492：PVSVMGIDLGVNPAFAYAVCT；
(2)664-763：KSYIDYYKNLRLDTLKKLTCAIVRTARSHGVEIVALEDIKRVDYDDQVKRAKENSLLSLWAPGMILERIEQELANEGIRTWRIDPRHTSQTACITDEFGY；
(3)778-800：GELLRVNSDVNAAINIARRFLTR。
实施例2.Cas-sf19蛋白在进行核酸检测时的应用
本实施例通过体外检测以验证Cas-sf19的trans切割活性。本实施例中利用可以与靶核酸配对的gRNA引导Cas-sf19蛋白识别并结合在靶核酸上；随后，Cas-sf19蛋白激发对任意单链核酸的trans切割活性，从而切割体系里的单链核酸检测器；单链核酸检测器的两端分别设置荧光基团和淬灭基团，如果单链核酸检测器被切割，则会激发荧光；在其他的实施方式中，单链核酸检测器的两端还可以设置成能够被胶体金检测的标记。
本实施例中选择靶核酸为单链DNA，N-B-i3g1-ssDNA0，其序列为：CGACATTCCGAAGAACGCTGAAGCGCTGGGGGCAAATTGTGCAATTTGCGGC；
gRNA序列为:GUGCUGCCGGUCUCUAAUCGGGGAUCGGAAUUGCAC CCCCCAGCGCUUCAGCG UUC(下划线区域为靶向区)；
单链核酸检测器序列为FAM-TTGTT-BHQ1；
采用如下反应体系：Cas-sf19终浓度为50nM，gRNA终浓度为50nM，靶核酸终浓度为50nM,单链核酸检测器终浓度200nM。37℃孵育，读取FAM荧光/1min。对照组不添加靶核酸。
如图2所示，与不加靶核酸的对照相比，在有靶核酸存在的情况下，Cas-sf19切割体系里的单链核酸检测，快速的报告出荧光。以上实验反映出，配合单链核酸检测器，Cas-sf19可以用于靶核酸的检测。图2中，1为添加靶核酸的实验结果，2为不添加靶核酸的对照组。另外，采用其他的单链核酸检测器，例如，5'6-FAM/TTATT/3'BHQ1，Cas-sf19同样可以表现出良好的切割活性，用于体外的核酸检测。
实施例3.Cas-sf19蛋白的PAM结构域鉴定
构建Cas-sf19蛋白表达质粒：对核酸序列进行大肠杆菌密码子优化后进行基因合成，连入大肠杆菌表达载体PeT28(a)+载体。在PeT28(a)+-Cas-sf19蛋白载体中加入JM23119启动子启动Cas-sf19CrRNA转录。形成载体：PeT28(a)+-Cas-sf19-JM23119-crRNA；PAM文库的构建：合成序列CGTGTTTCGTAAAGTCTGGAAACGCGGAAGCCCCCAGCGCTTCAGCGTTCNNNNNNTCCCCTACGTGCTGCTGAAGTTGCCCGCAA，N为随机脱氧核苷酸。经Klenow酶补平后连入pacyc184载体。转化大肠杆菌后提取质粒形成PAM文库。
PAM文库消减实验：制备感受态：BL21(DE3)-PeT28(a)+-Cas-sf19-JM23119-crRNA。PAM文库质粒转化感受态：BL21(DE3)-PeT28(a)+-Cas-sf19-JM23119-crRNA，涂布在含有卡那霉素、氯霉素的LB平板上，37℃过夜培养后收集菌体，大提试剂盒进行质粒提取，获得消减后PAM库。以30ng/μL质粒(PAM文库)为模板引物进行PCR反应获得对照组样品，以30ng/μL质粒(消减后PAM库)为模板进行PCR反应获得实验组样品。对照组样品、实验组样品送二代测序进行数据分析。
获得Cas-sf19的PAM结构：对于4096种PAM序列，分别统计实验组和对照组中出现的次数，并用各自组所有PAM序列总数进行标准化。对于每条PAM消耗水平的计算方式为log2(对照组标准化值/实验组标准化值)，当该值大于3.5时，认为这条PAM被显著消耗。然后使用Weblogo对显著消耗的PAM序列进行预测，发现PAM结构为ATG，如图3所示。
实施例4.Cas-sf19蛋白在进行双链靶核酸检测时的应用
本实施例通过体外检测以验证Cas-sf19在体外以双链DNA为靶点所激发的trans切割活性。本实施例中利用可以与靶核酸配对的gRNA引导Cas-sf19蛋白识别并结合在靶核酸上；随后，Cas-sf19蛋白激发对任意单链核酸的trans切割活性，从而切割体系里的单链核酸检测器；单链核酸检测器的两端分别设置荧光基团和淬灭基团，如果单链核酸检测器被切割，则会激发荧光；在其他的实施方式中，单链核酸检测器的两端还可以设置成能够被胶体金检测的标记。
本实施例中选择靶核酸为双链DNA(质粒)，T-N-B-ATG，其序列为：AACATTGGCCGCAAATTGCACAAT
Figure PCTCN2022075030-appb-000002
CCCCCAGCGCTTCAGCGTTCTTCGGAATGTCGCGCATTGGCATGGAAGTCACACCTTCGGGAACGTGGTTGACCTACACAGGTGCCATCAAATTGGATGACAAAGATCCAAATTTCA连入载体T-Vector-pEASY-Blunt Simple Cloning Vector；斜体部分(ATG)为PAM序列，下划线区域为靶向区。
gRNA序列为:GUGCUGCCGGUCUCUAAUCGGGGAUCGGAAUUGCAC CCCCCAGCGCUUCAGCG UUC(下划线区域为靶向区)；
单链核酸检测器序列为FAM-TTATT-BHQ1；
采用如下反应体系：Cas-sf19终浓度为100nM，gRNA终浓度为50nM，双链靶核酸终浓度为5ng/μL,单链核酸检测器终浓度200nM。37℃孵育，读取FAM荧光/30s。
结果如图4所示，与不加靶核酸的对照相比，在有靶核酸存在的情况下，Cas-sf19切割体系里的单链核酸检测，快速的报告出荧光。以上实验反映出，配合单链核酸检测器，Cas-sf19可以用于双链靶核酸的检测。图4中，1为添加双链靶核酸的实验结果，2为不添加双链靶核酸的对照组。
实施例5.Cas-sf19蛋白在进行核酸编辑时的应用
本实施例检测Cas-sf19的双链DNA的cis切割活性。本实施例中利用可以与靶核酸配对的gRNA引导Cas-sf19蛋白识别并结合在靶核酸上，从而切割体系里的靶核酸，切割后的靶核酸进行琼脂糖电泳检测。
本实施例中选择靶核酸为双链DNA(质粒)，T-N-B-ATG，其序列为：AACATTGGCCGCAAATTGCACAAT
Figure PCTCN2022075030-appb-000003
CCCCCAGCGCTTCAGCGTTCTTCGGAATGTCGCGCATTGGCATGGAAGT CACACCTTCGGGAACGTGGTTGACCTACACAGGTGCCATCAAATTGGATGACAAAGATCCAAATTTCA连入载体T-Vector-pEASY-Blunt Simple Cloning Vector；斜体部分为PAM序列，下划线区域为靶向区。gRNA序列为:GUGCUGCCGGUCUCUAAUCGGGGAUCGGAAUUGCAC CC CCCAGCGCUUCAGCGUUC(下划线区域为靶向区)；采用如下反应体系：Cas-sf19终浓度为100nM，gRNA终浓度为50nM，双链靶核酸终浓度为5ng/μL。37℃孵育1h。实验组添加gRNA和靶核酸，对照组不添加gRNA。
结果如图5所示，与不加gRNA的对照相比，实验组中的Cas-sf19能够切割体系里的双链核酸，表现出明显的切割条带。这表明Cas-sf19可以用于双链靶核酸的切割和编辑。图5中，1为实验组，2为对照组。
尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公布的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本发明的保护范围之内。本发明的全部分为由所附权利要求及其任何等同物给出。

Claims (19)

  1. 一种Cas蛋白，其特征在于，所述Cas蛋白为以下I-III任一所述的Cas蛋白：
    I、Cas蛋白的氨基酸序列与SEQ ID No.1相比，具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％的序列同一性，并且基本保留了SEQ ID No.1的生物学功能；
    II、所述Cas蛋白的氨基酸序列与SEQ ID No.1相比，具有一个或多个氨基酸的置换、缺失或添加的序列，并且基本保留了SEQ ID No.1的生物学功能；
    III、所述Cas蛋白包含SEQ ID No.1所示的氨基酸序列。
  2. 一种融合蛋白，所述融合蛋白包括权利要求1所述的Cas蛋白和其他的修饰部分。
  3. 一种分离的多核苷酸，其特征在于，所述多核苷酸为编码权利要求1所述Cas蛋白的多核苷酸序列，或编码权利要求2所述融合蛋白的多核苷酸序列。
  4. 一种gRNA，其特征在于，所述gRNA包括能够结合权利要求1所述的Cas蛋白的同向重复序列和能够靶向靶序列的引导序列。
  5. 一种同向重复序列，其特征在于，所述同向重复序列包含SEQ ID No.3所示的序列。
  6. 一种载体，其特征在于，所述载体包含权利要求3所述的多核苷酸以及与之可操作连接的调控元件。
  7. 一种CRISPR-Cas系统，其特征在于，所述系统包括权利要求1所述的Cas蛋白以及至少一种权利要求4所述的gRNA。
  8. 一种载体系统，其特征在于，所述载体系统包括一种或多种载体，该一种或多种载体包括：
    a)第一调控元件，该第一调控元件可操作地与权利要求4所述的gRNA连接，
    b)第二调控元件，该第二调控元件可操作地与权利要求1所述的Cas蛋白连接；
    其中组分(a)和(b)位于该系统的相同或不同载体上。
  9. 一种组合物，其特征在于，所述组合物包含：
    (i)蛋白组分，其选自：权利要求1所述的Cas蛋白或权利要求2所述的融合蛋白；
    (ii)核酸组分，其选自：权利要求4所述的gRNA，或编码权利要求4所述的gRNA的核酸，或权利要求4所述的gRNA的前体RNA，或编码权利要求4所述的gRNA的前体RNA核酸；
    所述蛋白组分与核酸组分相互结合形成复合物。
  10. 一种活化的CRISPR复合物，所述活化的CRISPR复合物包含：
    (i)蛋白组分，其选自：权利要求1所述的Cas蛋白或权利要求2所述的融合蛋白；
    (ii)核酸组分，其选自：权利要求4所述的gRNA，或编码权利要求4所述的gRNA的核酸，或权利要求4所述的gRNA的前体RNA，或编码权利要求4所述的gRNA的前体RNA核酸；
    (iii)结合在权利要求4所述的gRNA上的靶序列。
  11. 一种工程化的宿主细胞，其特征在于，所述宿主细胞包含权利要求1所述的Cas蛋白，或权利要求2所述的融合蛋白，或权利要求3所述的多核苷酸，或权利要求6所述的载体，或权利要求7所述的CRISPR-Cas系统，或权利要求8所述的载体系统，或权利要求9所述的组合物，或权利要求10所述的活化的CRISPR复合物。
  12. 权利要求1所述的Cas蛋白，或权利要求2所述的融合蛋白，或权利要求3所述的多核苷酸，或权利要求6所述的载体，或权利要求7所述的CRISPR-Cas系统，或权利要求8所述的载体系统，或权利要求9所述的组合物，或权利要求10所述的活化的CRISPR复合物，或权利要求11所述的宿主细胞在基因编辑、基因靶向或基因切割中的应用；或者，在制备用于基因编辑、基因靶向或基因切割的试剂或试剂盒中的用途。
  13. 权利要求1所述的Cas蛋白，或权利要求2所述的融合蛋白，或权利要求3所述的多核苷酸，或权利要求6所述的载体，或权利要求7所述的CRISPR-Cas系统，或权利要求8所述的载体系统，或权利要求9所述的组合物，或权利要求10所述的活化的CRISPR复合物，或权利要求11所述的宿主细胞在选自如下任一 或任意几种中的应用：
    靶向和/或编辑靶核酸；切割双链DNA、单链DNA或单链RNA；非特异性切割和/或降解侧枝核酸；非特异性的切割单链核酸；核酸检测；特异性地编辑双链核酸；碱基编辑双链核酸；碱基编辑单链核酸。
  14. 一种编辑靶核酸、靶向靶核酸或切割靶核酸的方法，所述方法包括将靶核酸与权利要求1所述的Cas蛋白，或权利要求2所述的融合蛋白，或权利要求3所述的多核苷酸，或权利要求6所述的载体，或权利要求7所述的CRISPR-Cas系统，或权利要求8所述的载体系统，或权利要求9所述的组合物，或权利要求10所述的活化的CRISPR复合物，或权利要求11所述的宿主细胞进行接触。
  15. 一种切割单链核酸的方法，所述方法包括，使核酸群体与权利要求1所述的Cas蛋白和权利要求4所述的gRNA接触，其中所述核酸群体包含靶核酸和至少一个非靶单链核酸，所述gRNA能够靶向所述靶核酸，所述Cas蛋白切割所述非靶单链核酸。
  16. 一种用于基因编辑、基因靶向或基因切割的试剂盒，所述试剂盒包括权利要求1所述的Cas蛋白，或权利要求2所述的融合蛋白，或权利要求3所述的多核苷酸，或权利要求6所述的载体，或权利要求7所述的CRISPR-Cas系统，或权利要求8所述的载体系统，或权利要求9所述的组合物，或权利要求10所述的活化的CRISPR复合物，或权利要求11所述的宿主细胞。
  17. 一种用于检测样品中的靶核酸的试剂盒，所述试剂盒包含：(a)权利要求1所述的Cas蛋白，或编码所述Cas蛋白的核酸；(b)权利要求4所述的gRNA，或编码所述gRNA的核酸，或包含所述gRNA的前体RNA，或编码所述前体RNA的核酸；和(c)为单链的且不与所述gRNA杂交的单链核酸检测器。
  18. 权利要求1所述的Cas蛋白，或权利要求2所述的融合蛋白，或权利要求3所述的多核苷酸，或权利要求6所述的载体，或权利要求7所述的CRISPR-Cas系统，或权利要求8所述的载体系统，或权利要求9所述的组合物，或权利要求10所述的活化的CRISPR复合物，或权利要求11所述的宿主细胞在制备制剂或试剂盒中的用途，所述制剂或试剂盒用于：
    (i)基因或基因组编辑；
    (ii)靶核酸检测和/或诊断；
    (iii)编辑靶基因座中的靶序列来修饰生物或非人类生物；
    (iv)疾病的治疗；
    (v)靶向靶基因；
    (vi)切割目的基因。
  19. 一种检测样品中靶核酸的方法，所述方法包括将样品与权利要求1所述的Cas蛋白、gRNA(指导RNA)和单链核酸检测器接触，所述gRNA包括与所述Cas蛋白结合的区域和与靶核酸杂交的指导序列；检测由所述Cas蛋白切割单链核酸检测器产生的可检测信号，从而检测靶核酸；所述单链核酸检测器不与所述gRNA杂交。
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116004573A (zh) * 2022-10-25 2023-04-25 山东舜丰生物科技有限公司 编辑活性提高的Cas蛋白及其应用
CN116286742A (zh) * 2022-09-29 2023-06-23 隆平生物技术(海南)有限公司 CasD蛋白、CRISPR/CasD基因编辑系统及其在植物基因编辑中的应用
WO2024041299A1 (zh) * 2022-08-25 2024-02-29 山东舜丰生物科技有限公司 突变的CRISPR-Cas蛋白及其应用
CN117821424A (zh) * 2023-03-07 2024-04-05 山东舜丰生物科技有限公司 一种优化的IscB蛋白及其应用

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115725543A (zh) * 2022-10-25 2023-03-03 山东舜丰生物科技有限公司 Crispr酶以及系统
CN116355878B (zh) * 2023-02-28 2024-04-26 华中农业大学 新型TnpB编程性核酸酶及其应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108513582A (zh) * 2015-06-18 2018-09-07 布罗德研究所有限公司 新型crispr酶以及系统
CN111304180A (zh) * 2019-06-04 2020-06-19 山东舜丰生物科技有限公司 一种新的dna核酸切割酶及其应用
CN111770992A (zh) * 2018-11-15 2020-10-13 中国农业大学 CRISPR-Cas12j酶和系统

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050181324A1 (en) * 2004-02-13 2005-08-18 Hare Robert V. Polyorganosiloxane dental impression materials with improved wetting and stability
US11453874B2 (en) * 2017-06-07 2022-09-27 The Rockefeller University Enhancement of CRISPR gene editing or target destruction by co-expression of heterologous DNA repair protein
EP3740580A4 (en) * 2018-01-19 2021-10-20 Duke University GENOME ENGINEERING WITH CRISPR-CAS SYSTEMS IN EUKARYONTS
CN110684755B (zh) * 2018-07-05 2021-12-31 清华大学 基于进化信息构建嵌合SaCas9用于增强和扩展PAM位点的识别
JP7216877B2 (ja) * 2018-10-29 2023-02-02 中国▲農▼▲業▼大学 新規なＣＲＩＳＰＲ／Ｃａｓ１２ｆ酵素およびシステム
CN111690717B (zh) * 2020-04-30 2023-05-30 山东舜丰生物科技有限公司 基于crispr技术进行目标核酸检测的方法和系统
CN111996236B (zh) * 2020-05-29 2021-06-29 山东舜丰生物科技有限公司 基于crispr技术进行靶核酸检测的方法
CN111690773B (zh) * 2020-06-17 2021-08-20 山东舜丰生物科技有限公司 利用新型Cas酶进行目标核酸检测的方法和系统

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108513582A (zh) * 2015-06-18 2018-09-07 布罗德研究所有限公司 新型crispr酶以及系统
CN111770992A (zh) * 2018-11-15 2020-10-13 中国农业大学 CRISPR-Cas12j酶和系统
CN111304180A (zh) * 2019-06-04 2020-06-19 山东舜丰生物科技有限公司 一种新的dna核酸切割酶及其应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
MAKAROVA KIRA S.; WOLF YURI I.; IRANZO JAIME; SHMAKOV SERGEY A.; ALKHNBASHI OMER S.; BROUNS STAN J. J.; CHARPENTIER EMMANUELLE; CH: "Evolutionary classification of CRISPR–Cas systems: a burst of class 2 and derived variants", NATURE REVIEWS MICROBIOLOGY, vol. 18, no. 2, 19 December 2019 (2019-12-19), GB , pages 67 - 83, XP036990744, ISSN: 1740-1526, ISBN: NATURE PUBLISHING GROUP, GB, DOI: 10.1038/s41579-019-0299-x *
SAFARI FATEMEH, ZARE KHADIJEH, NEGAHDARIPOUR MANICA, BAREKATI-MOWAHED MAZYAR, GHASEMI YOUNES: "CRISPR Cpf1 proteins: structure, function and implications for genome editing", CELL & BIOSCIENCE, vol. 9, no. 1, 1 December 2019 (2019-12-01), XP055781539, DOI: 10.1186/s13578-019-0298-7 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024041299A1 (zh) * 2022-08-25 2024-02-29 山东舜丰生物科技有限公司 突变的CRISPR-Cas蛋白及其应用
CN116286742A (zh) * 2022-09-29 2023-06-23 隆平生物技术(海南)有限公司 CasD蛋白、CRISPR/CasD基因编辑系统及其在植物基因编辑中的应用
CN116286742B (zh) * 2022-09-29 2023-11-17 隆平生物技术(海南)有限公司 CasD蛋白、CRISPR/CasD基因编辑系统及其在植物基因编辑中的应用
CN116004573A (zh) * 2022-10-25 2023-04-25 山东舜丰生物科技有限公司 编辑活性提高的Cas蛋白及其应用
CN116004573B (zh) * 2022-10-25 2023-09-12 山东舜丰生物科技有限公司 编辑活性提高的Cas蛋白及其应用
WO2024087405A1 (zh) * 2022-10-25 2024-05-02 山东舜丰生物科技有限公司 编辑活性提高的Cas蛋白及其应用
CN117821424A (zh) * 2023-03-07 2024-04-05 山东舜丰生物科技有限公司 一种优化的IscB蛋白及其应用

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