WO2024040874A1 - Mutated cas12j protein and use thereof - Google Patents

Mutated cas12j protein and use thereof Download PDF

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WO2024040874A1
WO2024040874A1 PCT/CN2023/074379 CN2023074379W WO2024040874A1 WO 2024040874 A1 WO2024040874 A1 WO 2024040874A1 CN 2023074379 W CN2023074379 W CN 2023074379W WO 2024040874 A1 WO2024040874 A1 WO 2024040874A1
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protein
cas
nucleic acid
sequence
target
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Chinese (zh)
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段志强
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山东舜丰生物科技有限公司
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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
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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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8205—Agrobacterium mediated transformation
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • 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]
    • C—CHEMISTRY; METALLURGY
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    • C12N2510/00—Genetically modified cells
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the field of gene editing, in particular to the field of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology. Specifically, the present invention relates to a mutated Cas12j protein with increased activity and its application.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • CRISPR/Cas technology is a widely used gene editing technology. It uses RNA guidance to specifically bind to target sequences on the genome and cut DNA to produce double-stranded breaks. It 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. It recognizes the PAM motif of 3’-NGG and performs blunt-end cleavage of the target sequence.
  • the CRISPR/Cas Type V system is a newly discovered CRISPR system that has a 5’-TTN motif and performs sticky end cleavage of target sequences, such as Cpf1, C2c1, CasX, and CasY.
  • target sequences such as Cpf1, C2c1, CasX, and CasY.
  • the different CRISPR/Cas currently existing have different advantages and disadvantages. For example, Cas9, C2c1 and CasX all require two RNAs for guide RNA, while Cpf1 only requires 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 relatively 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.
  • Chinese invention patent CN111770992B discloses a Cas protein, Cas12j.19, and also discloses that this protein can perform gene editing in eukaryotic cells. However, its editing activity is not high. In order to improve the editing efficiency of this protein, this application The protein was optimized to improve its editing efficiency in eukaryotic cells.
  • the present invention provides a Cas mutant protein with improved activity or increased activity.
  • the mutant protein has at the following amino acid positions corresponding to the amino acid sequence shown in SEQ ID No. 1 There is a mutation at position 100.
  • the amino acid at position 100 is mutated to an amino acid other than E, for example, A, V, G, L, D, F, W, Y, N, S, Q, K, M, T, C , P, H, R, I; preferably, the mutation is K, Y, M, F, R, W, S, H, V, I, N, C or L.
  • the amino acid sequence of the parent Cas protein has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% compared to SEQ ID No. 1 , at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity.
  • the parent Cas protein is a Cas protein of the Cas12 family, preferably a Cas protein of the Cas12j family, for example, Cas12j.3-Cas12j.22 disclosed in CN111770992B, etc.; in a preferred embodiment, the parent Cas protein Cas protein is Cas12j19; the amino acid sequence of Cas12j19 has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% compared with SEQ ID No.1 %, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, At least 99.8%, or at least 99.9%, or 100% sequence identity.
  • the Cas mutant protein is selected from any group of I-III below:
  • Cas mutant protein obtained by mutating the amino acid sequence shown in SEQ ID No. 1 at the following amino acid positions: position 100; preferably, the amino acid at position 100 is mutated to a non-E amino acid, for example, A , V, G, L, D, F, W, Y, N, S, Q, K, M, T, C, P, H, R, I; preferably, the mutation is K, Y, M, F, R , W, S, H, V, I, N, C or L;
  • the Cas mutant protein described in I Compared with the Cas mutant protein described in I, it has the mutation site described in I; and, compared with the Cas mutant protein described in I, it has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4% , a Cas mutant protein with at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity;
  • the Cas mutant protein described in I has the mutation site described in I; and, compared with the Cas mutant protein described in I, it has a sequence with one or more amino acid substitutions, deletions or additions. ;
  • the one or more amino acids include substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids.
  • Cas proteins or Cas12j proteins from various organisms can be used as parent Cas proteins, and in some embodiments, the parent Cas proteins or Cas12j proteins have nuclease activity.
  • the parent Cas protein is a nuclease that cleaves both strands of a target duplex nucleic acid (eg, duplex DNA).
  • the parent Cas protein is a nickase, i.e., cleaves a single strand of a target duplex nucleic acid (eg, duplex DNA).
  • the parent Cas protein is a natural wild-type Cas protein; in other embodiments, the parent Cas protein is an engineered Cas protein.
  • the structure of a protein can be changed without adversely affecting its activity and functionality.
  • one or more conservative amino acid substitutions can be introduced in the amino acid sequence of the protein without affecting the activity and/or functionality of the protein molecule. Or adversely affect the three-dimensional structure. Examples and implementations of conservative amino acid substitutions will be apparent to those skilled in the art.
  • the amino acid residue can be replaced with another amino acid residue belonging to the same group as the site to be replaced, that is, a non-polar amino acid residue can be substituted for another non-polar amino acid residue, and a polar uncharged amino acid residue can be substituted.
  • amino acid residue is substituted for another polar uncharged amino acid residue
  • a basic amino acid residue is substituted for another basic amino acid residue
  • an acidic amino acid residue is substituted for another acidic amino acid residue.
  • Such substituted amino acid residues may or may not be composed of Encoded by the genetic code.
  • Conservative substitutions in which one amino acid is replaced by another amino acid belonging to the same group fall within the scope of the invention as long as the substitution does not result in inactivation of the biological activity of the protein. Therefore, the protein of the present invention may contain one or more conservative substitutions in the amino acid sequence, and these conservative substitutions are preferably produced by substitutions according to Table 1.
  • the invention also encompasses proteins that also contain one or more other non-conservative substitutions, as long as the non-conservative substitutions do not significantly affect the desired function and biological activity of the protein of the invention.
  • Non-essential amino acid residues are amino acid residues that can be altered (deletion, substitution or replacement) without altering biological activity, whereas "essential” amino acid residues are required for biological activity.
  • a “conservative amino acid substitution” is a substitution in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Amino acid substitutions can be made in non-conserved regions of the above-mentioned Cas mutant proteins. 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 fewer conservative or non-conservative changes in conserved regions.
  • proteins that change one or more amino acid residues from the N and/or C terminus of the 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, which include PCR amplification that alters or extends the protein coding sequence by including the amino acid coding sequence among the oligonucleotides used in the PCR amplification.
  • proteins can be altered in a variety of ways, including amino acid substitutions, deletions, truncation and insertions, and methods for such manipulations are generally known in the art.
  • amino acid sequence variants of the above-mentioned proteins can be prepared by mutating DNA. This can also be accomplished by other forms of mutagenesis and/or by directed evolution, for example using known mutagenesis, recombination and/or shuffling methods in combination with relevant screening methods for single or multiple amino acids. Acid substitutions, deletions and/or insertions.
  • these minor amino acid changes in the Cas proteins of the invention can occur (eg, naturally occurring mutations) or be 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 domains of the protein, the properties of the polypeptide may be altered, but the polypeptide may maintain its activity. If mutations are present that are not close to the catalytic domain, active site, or other functional domains, smaller effects can be expected.
  • the catalytic domain, active site or other functional domains of a protein can also be determined by physical analysis of the structure, such as through techniques such as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, combined with putative key sites point amino acid mutations.
  • amino acid residues can be represented by single letters or three letters, for example: alanine (Ala, A), valine (Val, V), glycine (Gly, G), leucine (Leu, L), glutamic acid (Gln, Q), phenylalanine (Phe, F), tryptophan (Trp, W), tyrosine (Tyr, Y), aspartic acid (Asp, D), asparagine (Asn, N), glutamic acid (Glu, E), lysine (Lys, K), methionine (Met, M), serine (Ser, S), threonine (Thr, T), cysteine (Cys, C), proline (Pro, P), isoleucine (Ile, I), histidine (His, H), arginine (Arg, R).
  • AxxB means that amino acid A at position xx is changed to amino acid B. Unless otherwise specified, amino acid A at position xx from the N-terminus is changed to amino acid B.
  • E100R means that the E at position 100 is mutated into an R.
  • mutations exist at multiple amino acid sites at the same time they can be expressed in a similar form to E328R-N369R.
  • E328R-N369R represents the mutation of E to R at position 328 and N to R at position 369.
  • the specific amino acid position (numbering) within the protein of the present invention is determined by comparing the amino acid sequence of the target protein with SEQ ID No. 1 using standard sequence alignment tools, such as using the Smith-Waterman algorithm or using CLUSTALW2 The algorithm aligns two sequences, with the sequences considered to be aligned when the alignment score is highest. Alignment scores can be calculated according to the method described in Wilbur, W.J. and Lipman, D.J. (1983) Rapid similarity searches of nucleic acid and protein data banks. Proc. Natl. Acad. Sci. USA, 80: 726-730.
  • the amino acid sequence of the parent Cas protein has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% compared to SEQ ID No. 1 , at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity.
  • the Cas mutant protein is selected from any group of I-III below:
  • a Cas mutant protein obtained by mutating the amino acid sequence shown in SEQ ID No. 1 at any one or several of the following amino acid positions: position 100;
  • the Cas mutant protein described in I Compared with the Cas mutant protein described in I, it has the mutation site described in I; and, compared with the Cas mutant protein described in I, it has at least 80%, at least 85%, at least 90%, at least A Cas mutant protein with 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity;
  • the Cas mutant protein described in I has the mutation site described in I; and, compared with the Cas mutant protein described in I, it has a sequence with one or more amino acid substitutions, deletions or additions. ; the one or more Amino acids include substitutions, deletions, or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.
  • the biological functions of the Cas protein include, but are not limited to, the activity of binding to guide RNA, endonuclease activity, and the activity of binding to and cutting specific sites in the target sequence under the guidance of guide RNA, including but not limited to Cis cleavage activity. and Trans cleavage activity.
  • Cas mutant protein can also be called a mutated Cas protein or a Cas protein variant.
  • the present invention also provides a fusion protein, which includes the above-mentioned Cas mutant protein and other modified parts.
  • the modifying moiety is selected from another protein or polypeptide, a detectable label, or any combination thereof.
  • the modified moiety is selected from an epitope tag, a reporter gene sequence, a nuclear localization signal (NLS) sequence, a targeting moiety, a transcriptional activation domain (e.g., VP64), a transcriptional repression domain (e.g., KRAB domain or SID domain), a nuclease domain (e.g., Fok1), and a domain with an activity selected from: nucleotide deaminase, adenosine deaminase, cytidine deaminase, methyl enzyme activity, demethylase, transcription activation activity, transcription repression 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 any combination thereof.
  • the NLS sequence is well known to
  • the NLS sequence is located at, near or close to the end (eg, N-terminus, C-terminus or both ends) of the Cas protein of the invention.
  • the 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 Tags (e.g., for purification, detection, or tracing).
  • the reporter gene sequence is well known to those skilled in the art, and examples thereof include but are not limited to GST, HRP, CAT, GFP, HcRed, DsRed, CFP, YFP, BFP, etc.
  • the fusion protein of the present invention includes 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, etc.
  • MBP maltose-binding protein
  • DBD DNA-binding domain
  • the fusion proteins of the 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 linked to the N-terminus or C-terminus of the Cas protein of the invention.
  • the modified portion is connected to the N-terminus or C-terminus of the Cas protein of the invention through a linker.
  • linkers are well known in the art and examples include, but are not limited to, those containing one or more (eg, 1, 2, 3, 4 or 5) amino acids (eg, Glu or Ser) or amino acid derivatives (such as Ahx, ⁇ -Ala, GABA or Ava) linkers, or PEG, etc.
  • the Cas protein, protein derivative or fusion protein of the present invention is not limited by its production method. For example, it can be produced by genetic engineering methods (recombinant technology) or chemical synthesis methods.
  • the invention provides an isolated polynucleotide comprising:
  • the nucleotide sequence is codon-optimized for expression in prokaryotic cells. exist In one embodiment, the nucleotide sequence is codon-optimized for expression in eukaryotic cells.
  • the cells are animal cells, eg, mammalian cells.
  • the cells are human cells.
  • the cells are plant cells, for example cells of cultivated plants (such as cassava, corn, sorghum, wheat or rice), algae, trees or vegetables.
  • cultivated plants such as cassava, corn, sorghum, wheat or rice
  • algae such as trees or vegetables.
  • the polynucleotide is preferably single-stranded or double-stranded.
  • gRNA Guide RNA
  • the present invention provides a gRNA, which includes a first segment and a second segment; the first segment is also called a “skeleton region”, “protein binding segment”, “protein binding segment” Sequence”, or “Direct Repeat (Direct Repeat) sequence”; the second segment is also called “targeting sequence for targeting nucleic acid” or “targeting segment for targeting nucleic acid”, or “targeting target” sequence's boot sequence”.
  • the first segment of the gRNA can interact with the Cas protein of the present invention, so that the Cas protein and the gRNA form a complex.
  • the first segment is a direct repeat sequence as described above.
  • the targeting sequence of the targeting nucleic acid or the targeting segment of the targeting nucleic acid of the invention comprises a nucleotide sequence complementary to a sequence in the target nucleic acid.
  • the targeting sequence or the targeting segment of the targeting nucleic acid of the invention interacts with the target nucleic acid in a sequence-specific manner through hybridization (ie, base pairing).
  • the targeting sequence of a targeting nucleic acid or the targeting segment of a targeting nucleic acid may be altered, or may be modified to hybridize to any desired sequence within the target nucleic acid.
  • the nucleic acid is selected from DNA or RNA.
  • the 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% (e.g., at least 65%, at least 70%, at least 75%, at least 80% , at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100%).
  • the "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 Cas protein).
  • the gRNA of the present invention guides its interacting Cas protein to the specific nucleotide sequence within the target nucleic acid through the action of the targeting sequence of the targeted nucleic acid.
  • the guide RNA includes 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 to the target sequence.
  • the gRNA of the present invention can form a complex with the Cas protein.
  • the present invention also provides a vector, which includes the above-mentioned Cas mutant protein, isolated nucleic acid molecule or polynucleotide; preferably, it also includes a regulatory element operably connected thereto.
  • the regulatory element is selected from one or more of the following group: enhancer, transposon, promoter, terminator, leader sequence, polyadenylation sequence, and marker gene.
  • the vector includes a cloning vector, an expression vector, a shuttle vector, and an integration vector.
  • the vectors included in the system are viral vectors (e.g., retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated vectors, and herpes simplex vectors), and may also be plasmids, viruses, cosmids, Phage and other types, which are well known to those skilled in the art.
  • viral vectors e.g., retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated vectors, and herpes simplex vectors
  • the invention provides an engineered non-naturally occurring vector system, or a CRISPR-Cas system, which system includes a Cas mutant protein or a nucleic acid sequence encoding the Cas mutant protein and a nucleic acid encoding one or more guide RNAs. .
  • the nucleic acid sequence encoding the Cas mutant protein and the nucleic acid encoding one or more guide RNAs are artificially synthesized.
  • the nucleic acid sequence encoding the Cas mutant protein and encoding one or more guides does not occur naturally 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 guide the Cas protein to the genomic locus of the DNA molecule of the one or more gene products.
  • the Cas protein Upon reaching the target sequence location, the target sequence is modified, edited, or cleaved, whereby the expression of the one or more gene products is altered or modified.
  • the cells of the present invention include one or more of animals, plants or microorganisms.
  • the Cas protein is codon-optimized for expression in cells.
  • the Cas protein directs cleavage of one or both strands at the target sequence location.
  • the present invention also provides an engineered non-naturally occurring vector system.
  • the vector system may include one or more vectors, and the one or more vectors include:
  • components (a) and (b) are located on the same or different carriers of the system.
  • the first and second regulatory elements include promoters (e.g., constitutive promoters or inducible promoters), enhancers (e.g., 35S promoter or 35S enhanced promoter), internal ribosome entry sites (IRES), and others.
  • Expression control elements eg, transcription termination signals, such as polyadenylation signals and polyU sequences).
  • the vector in the system is a viral vector (eg, retroviral vector, lentiviral vector, adenoviral vector, adeno-associated vector, and herpes simplex vector), and may also be a plasmid, virus, cosmid, phage and other types, which are well known to those skilled in the art.
  • a viral vector eg, retroviral vector, lentiviral vector, adenoviral vector, adeno-associated vector, and herpes simplex vector
  • plasmid virus, cosmid, phage 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, microbubbles, 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 is present within the cell. In one embodiment, the target sequence is present in the nucleus or cytoplasm (eg, organelle). In one embodiment, the cell is a eukaryotic cell. In other embodiments, the cell is a prokaryotic cell.
  • the Cas protein is linked to one or more NLS sequences.
  • the fusion protein contains one or more NLS sequences.
  • the NLS sequence is linked to the N-terminus or 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 include direct repeats and spacers capable of hybridizing to target nucleic acids.
  • the Cas protein is capable of binding the guide RNA and targeting a target nucleic acid sequence complementary to the spacer sequence.
  • Protein-nucleic acid complexes/compositions Protein-nucleic acid complexes/compositions
  • the invention provides a complex or composition comprising:
  • Protein component which is selected from: the above-mentioned Cas protein, derivatized protein or fusion protein, and any combination thereof;
  • a nucleic acid component 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 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, which includes: (1) a protein component selected from: the Cas protein, derivatized protein or fusion protein of the present invention, and any combination thereof; (2) gRNA, which includes (a) a guide sequence capable of hybridizing to the target sequence; and (b) a direct repeat sequence capable of binding to the Cas protein of the invention; and (3) binding to the gRNA target sequence.
  • the binding is binding to the target nucleic acid through a targeting sequence on the gRNA that targets the nucleic acid.
  • activated CRISPR complex refers to the complex in which Cas protein, gRNA and target nucleic acid are combined or modified in the CRISPR system.
  • the Cas protein and gRNA of the present invention can form a binary complex, which is activated when combined with a nucleic acid substrate to form an activated CRISPR complex.
  • the nucleic acid substrate and the spacer sequence (or so-called spacer sequence) in the gRNA are , complementary to the guide sequence that hybridizes to the target nucleic acid).
  • the gRNA's spacer sequence exactly matches the target substrate.
  • the spacer sequence of the gRNA matches a portion (contiguous or discontinuous) of the target substrate.
  • the activated CRISPR complex can exhibit side-branching nucleic acid cleavage activity, and the side-branching nucleic acid cleavage activity refers to the non-specific cleavage activity or random cleavage activity of single-stranded nucleic acids exhibited by the activated CRISPR complex. Cleavage activity is also called trans cleavage activity in the art.
  • the Cas protein, gRNA, fusion protein, nucleic acid molecule, vector, system, complex and composition 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, dendrimers Transfection, heat shock transfection, nucleofection, magnetofection, lipofection, puncture transfection, optical transfection, reagent-enhanced nucleic acid uptake, and via liposomes, immunoliposomes, virus particles, artificial viruses Delivery of body etc.
  • the present invention provides a delivery composition, which includes a delivery vector, and one or any several selected from the following: Cas protein, fusion protein, nucleic acid molecule, vector, system, Complexes 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 (e.g., replication-deficient retroviruses , lentivirus, adenovirus or adeno-associated virus).
  • viral vectors e.g., replication-deficient 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, which cell or cell line or their progeny contains: Cas protein, fusion protein, nucleic acid according to the invention Molecules, protein-nucleic acid complexes, activated CRISPR complexes, vectors, delivery compositions of the invention.
  • the cell is a prokaryotic cell.
  • the cell is a eukaryotic cell.
  • the cells are mammalian cells.
  • the cells are human cells.
  • the cell is a non-human mammalian cell, such as a non-human primate, bovine, ovine, porcine, canine, monkey, rabbit, rodent (eg, rat or mouse) cell.
  • the cell is a non-mammalian eukaryotic cell, such as a cell of a poultry bird (eg, chicken), fish, or crustacean (eg, clam, shrimp).
  • the cells are plant cells, such as those of a monocot or dicot or a cultivated plant or a food crop such as cassava, corn, sorghum, soybean, wheat, oat or rice, e.g. Algae, trees or production plants, fruits or vegetables (for example, trees such as citrus trees, nut trees; nightshades, cotton, tobacco, tomatoes, grapes, coffee, cocoa, etc.).
  • a monocot or dicot or a cultivated plant or a food crop such as cassava, corn, sorghum, soybean, wheat, oat or rice, e.g. Algae, trees or production plants, fruits or vegetables (for example, trees such as citrus trees, nut trees; nightshades, cotton, tobacco, tomatoes, grapes, coffee, cocoa, etc.).
  • the cells are stem cells or stem cell lines.
  • a host cell of the invention contains a genetic or genomic modification that is not present in its wild type.
  • the Cas mutant protein, nucleic acid, the above composition, the above CIRSPR/Cas system, the above vector system, the above delivery composition or the above activated CRISPR complex or the above host cell of the present invention can be used for any one or any several of the following purposes: target To and/or edit target nucleic acid; cleave double-stranded DNA, single-stranded DNA or single-stranded RNA; non-specific cleavage and/or degradation of side branch nucleic acids; non-specific cleavage of single-stranded nucleic acids; nucleic acid detection; detection of nucleic acids in target samples; specificity Base editing of double-stranded nucleic acids; base editing of double-stranded nucleic acids; base editing of single-stranded nucleic acids. In other embodiments, it can also be used to prepare reagents or kits for any one or several of the above uses.
  • the invention also provides the application of the above-mentioned Cas protein, nucleic acid, above-mentioned composition, above-mentioned CIRSPR/Cas system, above-mentioned vector system, above-mentioned delivery composition or above-mentioned activated CRISPR complex in gene editing, gene targeting or gene cutting; Alternatively, use in the preparation of reagents or kits for gene editing, gene targeting or gene cleavage.
  • the gene editing, gene targeting or gene cleavage is performed intracellularly and/or extracellularly.
  • the invention also provides a method for editing target nucleic acid, targeting target nucleic acid or cutting target nucleic acid.
  • the method includes combining the target nucleic acid with the above-mentioned Cas protein, nucleic acid, above-mentioned composition, above-mentioned CIRSPR/Cas system, above-mentioned vector system, The above delivery composition or the above activated CRISPR complex is contacted.
  • the method is editing, targeting, or cleaving a target nucleic acid intracellularly or extracellularly.
  • the gene editing or editing target nucleic acid includes modifying genes, knocking out genes, changing the expression of gene products, repairing mutations, and/or inserting polynucleotides, and 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 vector 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 cutting single-stranded nucleic acids, which method includes contacting a nucleic acid population with the above-mentioned Cas protein and gRNA, wherein the nucleic acid population includes 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 to the Cas protein.
  • the gRNA is capable of targeting the target nucleic acid.
  • the contact may be inside the 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 vector system, the above-mentioned delivery composition or the above-mentioned activated CRISPR complex in non-specific cutting of single-stranded nucleic acids.
  • the present invention also provides a kit for gene editing, gene targeting or gene cutting, which kit includes the above-mentioned Cas protein, gRNA, nucleic acid, the above-mentioned composition, the above-mentioned CIRSPR/Cas system, the above-mentioned The vector system, the above-mentioned delivery composition, the above-mentioned activated CRISPR complex or the above-mentioned host cell.
  • kit includes the above-mentioned Cas protein, gRNA, nucleic acid, the above-mentioned composition, the above-mentioned CIRSPR/Cas system, the above-mentioned The vector system, the above-mentioned delivery composition, the above-mentioned activated CRISPR complex or the above-mentioned host cell.
  • the present invention also provides a kit for detecting target nucleic acid in a sample, the kit comprising: (a) Cas protein, or nucleic acid encoding the Cas protein; (b) 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 RNA can be cleaved or processed into the mature guide RNA described above.
  • the invention provides the above-mentioned Cas protein, nucleic acid, above-mentioned composition, above-mentioned CIRSPR/Cas system, above-mentioned vector system, above-mentioned delivery composition, above-mentioned activated CRISPR complex or above-mentioned host cell in preparation preparations or kits Purpose, the preparation or kit is used for:
  • the above-mentioned gene or genome editing is performed within or outside the cell.
  • the target nucleic acid detection and/or diagnosis is performed in vitro.
  • the treatment of the disease is the treatment of a condition caused by a defect in a target sequence in a target locus.
  • the 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 Cas protein-binding region and the guide sequence for hybridizing with the target nucleic acid; detecting the detectable signal generated by the Cas protein cutting single-stranded nucleic acid detector, thereby detecting the target nucleic acid; the single-stranded nucleic acid detector does not hybridize with the gRNA .
  • the present invention also provides a method for specifically modifying a target nucleic acid.
  • the method includes: combining the target nucleic acid with the above-mentioned Cas protein, nucleic acid, above-mentioned composition, above-mentioned CIRSPR/Cas system, above-mentioned carrier system, and above-mentioned delivery composition. or contact with the activated CRISPR complex described above.
  • the specific modification can occur in vivo or in vitro.
  • the specific modification can occur intracellularly or extracellularly.
  • the cells are selected from prokaryotic or eukaryotic cells, for example, animal cells, plant cells, or microbial cells.
  • the modification refers to a break in the target sequence, such as a single/double strand break in DNA, or a single strand break in RNA.
  • the method further includes 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 Part of the copy is integrated into the target nucleic acid.
  • the modification further includes inserting an editing template (eg, exogenous nucleic acid) into the break.
  • an editing template eg, exogenous nucleic acid
  • the method further includes contacting an editing template with the target nucleic acid or delivering it to a cell containing 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 that includes one or more of the target gene Insertions, deletions, or substitutions of multiple nucleotides, and in other embodiments, the mutations result in one or more amino acid changes in the protein expressed from the gene containing the target sequence.
  • the present invention provides a method for detecting target nucleic acid in a sample, the method comprising combining the sample with the above-mentioned Cas protein, nucleic acid, above-mentioned composition, above-mentioned CIRSPR/Cas system, above-mentioned carrier system, above-mentioned delivery composition or
  • the above-mentioned activated CRISPR complex is in contact with the single-stranded nucleic acid detector; the detectable signal generated by the Cas protein cutting the single-stranded nucleic acid detector is detected, thereby detecting the target nucleic acid.
  • the target nucleic acid includes ribonucleotides or deoxyribonucleotides; includes single-stranded nucleic acid and double-stranded nucleic acid, such as single-stranded DNA, double-stranded DNA, single-stranded RNA, and double-stranded RNA.
  • the target nucleic acid is derived from samples such as viruses, bacteria, microorganisms, soil, water sources, human bodies, animals, plants, etc.
  • the target nucleic acid is a product enriched or amplified by PCR, NASBA, RPA, SDA, LAMP, HAD, NEAR, MDA, RCA, LCR, RAM and other methods.
  • the target nucleic acid is a viral nucleic acid, a bacterial nucleic acid, a specific nucleic acid related to a disease, such as a specific mutation site or SNP site or a nucleic acid that is different from a control;
  • the virus is a plant Viruses or animal viruses, for example, papillomavirus, hepatovirus, herpesvirus, adenovirus, poxvirus, parvovirus, coronavirus; preferably, 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 a matching degree of at least 50% 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 completely match the gRNA.
  • the detection method may include one or more gRNAs with different guide sequences targeting different target sequences.
  • the single-stranded nucleic acid detector includes but is not limited to single-stranded DNA, single-stranded RNA, DNA-RNA hybrids, nucleic acid analogs, base modifications, and single-stranded nucleic acid detection containing abase spacers.
  • nucleic acid analogs include but are not limited to: locked nucleic acid, bridged nucleic acid, morpholine nucleic acid, ethylene 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' sulfur RNA, and combinations thereof, including optional ribonucleotide or deoxyribonucleotide residues.
  • the detectable signal is achieved in the following ways: vision-based detection, sensor-based detection, color detection, fluorescence signal-based detection, gold nanoparticle-based detection, fluorescence polarization, colloidal phase change/dispersion, electrochemical Chemical detection and semiconductor-based detection.
  • the two ends of the single-stranded nucleic acid detector are respectively provided with fluorescent groups and quenching groups.
  • the fluorescent group is selected from one or more of FAM, FITC, VIC, JOE, TET, CY3, CY5, ROX, Texas Red or LC RED460;
  • the quenching group is selected from BHQ1, BHQ2, BHQ3 , Dabcy1 or Tamra, or any one of them.
  • the 5' end and 3' end of the single-stranded nucleic acid detector are respectively provided with different labeling molecules, and colloidal gold detection is used to detect the time before and after the single-stranded nucleic acid detector is cleaved by the Cas protein.
  • Colloidal gold test results after being cleaved by Cas protein; the single-stranded nucleic acid detector will show different color results on the colloidal gold detection line and quality control line before being cleaved by Cas protein and after being cleaved by Cas protein.
  • the method of detecting a target nucleic acid may further include 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 may further include using an RNA reporter nucleic acid and a DNA reporter nucleic acid on different channels (e.g., fluorescent colors), and by measuring 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 is used to determine the level of detectable signal, and the level of detectable signal is sampled based on combining (eg, using a minimum or product).
  • channels e.g., fluorescent colors
  • the target gene is present within the cell.
  • the cell is a prokaryotic cell.
  • the cell is a eukaryotic cell.
  • the cells are animal cells.
  • the cells are human cells.
  • the cells are plant cells, for example cells of cultivated plants (such as cassava, corn, sorghum, wheat or rice), algae, trees or vegetables.
  • cultivated plants such as cassava, corn, sorghum, wheat or rice
  • algae such as 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.
  • Nucleic acid cleavage or cleavage of nucleic acids herein includes: DNA or RNA cleavage in the target nucleic acid produced by the Cas enzyme described herein (Cis cleavage), DNA or RNA cleavage in side nucleic acid substrates (single-stranded nucleic acid substrates) ( That is, 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-Cas system
  • CRISPR system CRISPR system
  • CRISPR/Cas complex refers to a complex formed by binding of guide RNA (guide RNA) or mature crRNA to Cas protein, which includes a guide sequence that hybridizes to the target sequence and is Protein-bound direct repeats allow the complex to recognize and cleave polynucleotides that hybridize to the guide RNA or mature crRNA.
  • Guide RNA guideRNA, gRNA
  • a guide RNA may comprise, consist essentially of, or consist of direct repeats and a leader sequence.
  • a guide sequence is any polynucleotide sequence that has sufficient complementarity 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 public 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, e.g., 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) formation. Perfect complementarity is not required, as long as sufficient complementarity exists to cause hybridization and promote the formation of a CRISPR/Cas complex.
  • the target sequence can comprise any polynucleotide, such as DNA or RNA.
  • the target sequence is located intracellularly or extracellularly.
  • the target sequence is located in the nucleus or cytoplasm of the cell.
  • the target sequence may be located in an organelle of a eukaryotic cell such as a mitochondria or chloroplast.
  • the sequence or template that can be used for recombination into a target locus containing the target sequence is called an "editing template" or "editing polynucleotide” or "editing 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 endogenous or exogenous to a cell (eg, a eukaryotic cell).
  • the target polynucleotide may 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 useless DNA).
  • the target sequence should be associated with a protospacer adjacent motif (PAM).
  • PAM protospacer adjacent motif
  • the single-stranded nucleic acid detector of the present invention refers to a sequence containing 2-200 nucleotides, preferably, having 2-150 nucleotides. Nucleotides are preferably 3-100 nucleotides, preferably 3-30 nucleotides, preferably 4-20 nucleotides, and 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 reporting groups or labeling molecules. When it is in the initial state (that is, when it is not cleaved), it does not present a reporting signal. When the single-stranded nucleic acid detector is cleaved, it displays a reporting signal. A detectable signal is produced, that is, there is a detectable difference after cutting and before cutting.
  • 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 several of LC RED460
  • the quenching group is selected from one or any of several BHQ1, BHQ2, BHQ3, Dabcy1 or Tamra.
  • the single-stranded nucleic acid detector has a first molecule (such as FAM or FITC) connected to the 5' end and a second molecule (such as biotin) connected to the 3' end.
  • the reaction system containing a single-stranded nucleic acid detector is used in conjunction with a flow strip to detect target nucleic acids (preferably, colloidal gold detection method).
  • the flow strip is designed to have two capture lines.
  • the sample contact end (colloidal gold) is provided with an antibody that binds the first molecule (i.e., the first molecule antibody), and the first line (control line) contains an antibody that binds the third molecule.
  • An antibody with one molecule of antibody contains a second molecule of antibody (that is, a second molecule of antibody, such as avidin) that binds to the second molecule at the second line (test line).
  • a second molecule of antibody such as avidin
  • the first molecule of antibody binds to the first molecule and carries the cleaved or uncleaved oligonucleotide to the capture line.
  • the cleaved reporter will bind to the first molecule of antibody at the first capture line.
  • antibody, while the uncleaved reporter will bind a second antibody molecule at the second capture line.
  • the binding of reporter groups in each line will result in a strong readout/signal (eg color).
  • the invention relates to the use of a flow strip as described herein for detecting nucleic acids.
  • the invention relates to methods of detecting nucleic acids using flow strips as defined herein, such as (lateral) flow tests or (lateral) flow immunochromatographic assays.
  • the molecules in the single-stranded nucleic acid detector can be replaced with each other, or the position of the molecules can be changed. As long as the reporting principle is the same 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 acids to be detected.
  • the quantitative detection index can be quantified based on the signal strength of the reporter group, such as the luminescence intensity of the fluorescent group, or the width of the color band.
  • wild type has the meaning commonly understood by those skilled in the art to mean the typical form of an organism, strain, gene, or characteristics that distinguish it from mutant or variant forms as it occurs in nature, It is isolated from sources in nature and has not been intentionally modified by man.
  • derivatization refers to the chemical modification of an amino acid, polypeptide, or protein to which one or more substituents have been covalently linked. Substituents may also be called side chains.
  • a derivatized protein is a derivative of the protein.
  • derivatization of the protein does not adversely affect the desired activity of the protein (e.g., binding activity to guide RNA, endonuclease activity, binding to a target sequence under the guidance of guide RNA) Specific site binding and cleavage activity), that is to say, the protein derivatives have the same activity as the protein.
  • protein derivatives refers to modified forms of proteins, for example, in which one or more amino acids of the protein can 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 mean the involvement of man. When these terms are used to describe a nucleic acid molecule or polypeptide, it is meant that the nucleic acid molecule or polypeptide is at least substantially free from at least one other component with which it is 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 similar function as the protein of which it is an 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 (for example, a position in each of two DNA molecules is occupied by adenine, or two A certain position in each polypeptide is occupied by lysine)
  • Percent identity between two sequences is a function of the number of matching positions common to the two sequences divided by the number of positions compared ⁇ 100. For example, if 6 out of 10 positions of two sequences match, then the two sequences are 60% identical.
  • the DNA sequences CTGACT and CAGGTT share 50% identity (matching at 3 positions out of a total of 6 positions).
  • comparisons are made when two sequences are aligned to yield maximum identity.
  • 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.). It is also possible to use the PAM120 weight residue table using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)) integrated into the ALIGN program (version 2.0).
  • the Needleman and Wunsch (J MoI Biol. 48:444-453 (1970)) algorithm can be used using the Blossum 62 matrix or PAM250 matrix with a gap weight of 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1, 2, 3, 4, 5 or 6 to determine the 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 including one or more free ends, no free ends (eg, circular); including DNA, RNA, or both. Nucleic acid molecules; and a wide variety of other polynucleotides known in the art.
  • the vector can be introduced into the host cell through transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell.
  • a vector can be introduced into a host cell to thereby produce a transcript, protein, or peptide, including from a protein, fusion protein, isolated nucleic acid molecule, etc. as described herein (e.g., a CRISPR transcript, such as a nucleic acid transcript , protein, or enzyme).
  • a vector can contain a variety of expression-controlling elements, 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 circle into which additional DNA segments can be inserted, such as by standard molecular cloning techniques.
  • viral vector in which virus-derived DNA or RNA sequences are present in packaging viruses (e.g., retroviruses, replication-deficient retroviruses, adenoviruses, replication-deficient adenoviruses, and adeno-associated virus) vector.
  • packaging viruses e.g., retroviruses, replication-deficient retroviruses, adenoviruses, replication-deficient adenoviruses, and adeno-associated virus
  • Viral vectors also contain polynucleotides carried by the virus 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, which includes, but is 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 (e.g., transcription termination signals, such as polyadenylation signals and PolyU sequence), for detailed description please refer to Goeddel, "GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY” 185, Academic Press, San Diego , California (1990).
  • regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host cells as well as those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences).
  • Tissue-specific promoters may primarily direct expression in the desired tissue of interest, such as muscle, neurons, bone, skin, blood, a specific organ (e.g., liver, pancreas), or a specific cell type (e.g., 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; the R-U 5' fragment in the LTR of HTLV-I ((Mol. Cell. Biol., pp. 8( 1), pp. 466-472, 1988); the SV40 enhancer; and the intron sequence between exons 2 and 3 of rabbit ⁇ -globin (Proc. Natl. Acad. Sci. USA., Volume 78(3), pages 1527-31, 1981).
  • promoter has a meaning known to those skilled in the art, which refers to a non-coding nucleotide sequence located upstream of a gene that can initiate the expression of a downstream gene.
  • a constitutive promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining the gene product, results in the gene product in the cell under most or all physiological conditions of the cell. of production.
  • An inducible promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, results in essentially only when an inducer corresponding to said promoter is present in the cell. The gene product is produced within the cell.
  • a tissue-specific promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, results in essentially only cells of the tissue type to which the promoter corresponds. Gene products are produced in cells.
  • a “nuclear localization signal” or “nuclear localization sequence” is an amino acid sequence that "tags" a protein for import into the nucleus via nuclear transport, ie, proteins with an NLS are transported to the nucleus. Typically, NLS contain positively charged Lys or Arg residues exposed on the protein surface. Exemplary nuclear localization sequences include, but are not limited to, NLS from: SV40 large T antigen, EGL-13, c-Myc, and TUS proteins.
  • the NLS includes the PKKKRKV sequence.
  • the NLS includes the sequence AVKRPAATKKAGQAKKKKLD.
  • the NLS includes the PAAKRVKLD sequence.
  • the NLS includes the sequence MSRRRKANPTKLSENAKKLAKEVEN. In some embodiments, the NLS includes the KLKIKRPVK sequence.
  • Other nuclear localization sequences include, but are not limited to, the acidic M9 domain of hnRNP A1, the sequences KIPIK and PY-NLS in the yeast transcriptional repressor Mat ⁇ 2.
  • operably linked is intended to mean that a 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 traditional Watson-Crick or other non-traditional types. Percent complementarity represents the percentage of residues (e.g., out of 10) in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence. 5, 6, 7, 8, 9, and 10 of them are 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Perfectly complementary” means that all contiguous residues of one nucleic acid sequence form hydrogen bonds with the same number of contiguous residues of a second nucleic acid sequence.
  • substantially complementary means that in a system having 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% complementarity, or 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 primarily to the target sequence and does not substantially hybridize to non-target sequences. Stringent conditions are often sequence dependent and vary depending on many factors. In general, the longer the sequence, the higher the temperature at which the sequence will hybridize specifically to its target sequence.
  • hybrid or “complementary” or “substantially complementary” means that a nucleic acid (e.g., RNA, DNA) contains nucleotide sequences that enable it to bind non-covalently, i.e., in a sequence-specific, antiparallel manner ( That is, a nucleic acid specifically binds a complementary nucleic acid) to form base pairs and/or G/U base pairs, “annealing” or “hybridization” with another nucleic acid.
  • a nucleic acid e.g., RNA, DNA
  • Hybridization requires that the two nucleic acids contain complementary sequences, although mismatches between bases may exist. 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 (e.g., 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 does not need to be 100% complementary to the sequence of its target nucleic acid in order to hybridize specifically.
  • the polynucleotide may comprise 60% or higher, 65% or higher, 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or Higher, 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 and form a complex; or there are at least 12, 15, 16, 17, 18, 19, 20 nucleic acid sequences representing the target sequence and gRNA. One, 21, 22 or more bases can complement each other and hybridize to form a complex.
  • the term "expression” refers to the process by which a DNA template is transcribed into a polynucleotide (eg, into mRNA or other RNA transcript) and/or the transcribed mRNA is subsequently translated into a peptide, The process of polypeptide or protein. Transcripts and encoded polypeptides may collectively be 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 multiple amino acid residues linked by peptide bonds.
  • the linker of the present invention can be a synthetic amino acid sequence or a naturally occurring polypeptide sequence, such as a polypeptide with hinge region function.
  • Such linker polypeptides are well known in the art (see, e.g., Holliger, P. et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R.J. et al. (1994) Structure 2:1121- 1123).
  • treating means treating or curing a condition, delaying the onset of symptoms of a condition, and/or delaying the progression of a condition.
  • the term "subject” includes, but is not limited to, various animals, plants, and microorganisms.
  • mammals such as bovines, equids, ovines, porcines, canines, felines, leporids, rodents (e.g., mice or rats), non-human primates animal (e.g., macaque or cynomolgus monkey) or human.
  • rodents e.g., mice or rats
  • non-human primates animal e.g., macaque or cynomolgus monkey
  • the subject eg, a human
  • has a disorder eg, a disorder resulting from a defect in a disease-associated gene.
  • plant is understood to mean any differentiated multicellular organism capable of photosynthesis and includes crop plants at any stage of maturity or development, in particular monocots or dicots, vegetable crops including artichokes, kohlrabi, Arugula, leeks, asparagus, lettuce (e.g., head, leaf, romaine), bok choy, yam, melons (e.g., melon, watermelon, crenshaw ), honeydew, romaine melon), rapeseed crops (e.g.
  • the present invention improves the activity of Cas12j19 protein through mutation and has broad application prospects.
  • FIG. 1 Verification results of the editing efficiency of mutated E100R protein and wild-type Cas12j19 in soybean.
  • the abscissa represents different target positions, and the ordinate represents editing efficiency.
  • Cas12i19 For the known Cas protein (Cas12j.19 in CN111770992B, in this example, it is called Cas12i19), the applicant used bioinformatics to predict the key amino acid positions that may affect its biological function, and assigned the amino acid positions to Point mutations were performed to obtain Cas mutant proteins with improved editing activity. Specifically, the Cas12j19 coding sequence was codon optimized and synthesized. The amino acid sequence of wild-type Cas12j19 is shown in SEQ ID No. 1, and its nucleic acid sequence is shown in SEQ ID No. 2. The potential Cas12j19 was analyzed through bioinformatics methods. Site-directed mutagenesis of amino acids that bind to the target sequence.
  • Variants of Cas proteins were generated by PCR-based site-directed mutagenesis.
  • the specific method is to divide the DNA sequence design of Cas12j19 protein into two parts centered on the mutation site, design two pairs of primers to amplify the two parts of the DNA sequence respectively, and introduce the sequence that needs to be mutated on the primers, and finally use Gibson cloning. Load both fragments into pcDNA3.3-eGFP vector.
  • the combination of mutants is constructed by splitting the DNA of the Cas12j19 protein into multiple segments and using PCR and Gibson clone.
  • Fragment amplification kit TransStart FastPfu DNA Polymerase (containing 2.5mM dNTPs), please refer to the instruction manual for specific experimental procedures.
  • Glue recovery kit Gel DNA Extraction Mini Kit, please refer to the instruction manual for detailed experimental procedures.
  • the kit used for vector construction pEASY-Basic Seamless Cloning and Assembly Kit (CU201-03). Please refer to the instruction manual for specific experimental procedures.
  • the mutated amino acid sites involved and the primer sequences used are shown in the following table:
  • the wild-type protein (WT) of Cas12j19 and the proteins with mutations in the above amino acid single sites (named after the mutation type) were obtained: D50R, E100R and N103D, which are relative to SEQ ID No.1
  • D50R, E100R and N103D which are relative to SEQ ID No.1
  • the 50th amino acid from the N terminus is mutated to R
  • the 100th amino acid from the N terminus is mutated to R
  • the 103rd amino acid from the N terminus is mutated to D.
  • Example 1 Different Cas proteins obtained in Example 1 were used to verify their gene editing activity in animal cells, and the target was designed for the Chinese hamster ovary cell (CHO) FUT8 gene, FUT8-3: ATGGAGGCTGTCTACAATGGGG A, the italicized part is the PAM sequence, and the remaining regions is the target area.
  • the vector pcDNA3.3 was modified to contain EGFP fluorescent protein and PuroR resistance gene.
  • the SV40NLS-Cas-XX fusion protein was inserted through the restriction site XbaI and PstI; the U6 promoter and gRNA sequence were inserted through the restriction site Mfe1.
  • the CMV promoter drives the expression of the fusion protein SV40NLS-Ca s-XX-NLS-GFP.
  • the protein Cas-XX-NLS and the protein GFP are connected using the connecting peptide T2A.
  • Promoter EF-1 ⁇ drives puromycin resistance gene expression.
  • Plating Plate the CHO cells until the confluence is 70-80%. The number of cells seeded in the 12-well plate is 8*10 ⁇ 4 cells/well.
  • Transfection Plate for 24 hours for transfection. Add 6.25 ⁇ l Hieff Trans TM liposome nucleic acid transfection reagent to 100 ⁇ l opti-MEM and mix well. Add 2.5ug plasmid to 100 ⁇ l opti-MEM and mix well.
  • Extract DNA, PCR amplify near the editing area, and send to hiTOM for sequencing cells are collected after trypsin digestion, and genomic DNA is extracted using a cell/tissue genomic DNA extraction kit (Biotech). Amplify the region near the target site from genomic DNA. PCR products were subjected to hiTOM sequencing. Sequencing data analysis, counting the sequence types and proportions within the range of 15nt upstream and 10nt downstream of the target position, counting the sequences with SNV frequency greater than/equal to 1% or non-SNV mutation frequency greater than/equal to 0.06%, to obtain Cas-XX The editing efficiency of the protein at the target position.
  • CHO cell F UT8 gene target sequence FUT8-Cas-XX-g3: ATGGAGGCTGTCTACAATGGGGA, the italicized part is the PAM sequence, and the rest of the region is the targeting region.
  • the gRNA sequence is: The bolded region is the targeting region, and the other regions are DR (direct repeat sequence) regions.
  • Figure 1 shows the editing activity of wild-type Cas12j19 protein (WT, shown in SEQ ID No. 1) and mutant proteins with single amino acid site mutations. As shown in Figure 1, compared with WT, mutations at the E100R amino acid site can significantly improve the editing efficiency of Cas12j19 protein, while mutations in D50R and N103D will reduce the editing efficiency of Cas12j19 protein.
  • Targets were designed for the FAD gene and BADH gene in the soybean genome.
  • the target information is shown in Table 2.
  • the designed targets were constructed into vectors containing the Cas mutant protein E100R in Example 1, which were used as verification vectors. With high editing efficiency, the constructed vector was used for transient transformation of soybean seedlings with Agrobacterium rhizogenes.
  • Soybean seed germination Mix nutrient soil and vermiculite in a ratio of 2:1, spread it flat on the plug tray, and place the soybean seeds. Sow the seeds into the plug tray at a depth of 0.5cm, pour an appropriate amount of water to prepare for germination.
  • Agrobacterium rhizogenes infects soybeans Use a blade to cut an inclined wound 1cm from the hypocotyls of the cotyledons of soybean seedlings that have grown for 7-14 days, and dip the wound into the wound to inoculate it with Agrobacterium rhizogenes.
  • the hypocotyls of the inoculated seedlings were then directly planted in a humid sterile vermiculite culture box, and each plant was poured with 10 ml of K599 infection suspension, and then maintained at high humidity for 16 days to promote soybean hairy root growth.
  • the DNA of the soybean hairy roots is extracted using the TPS method, and the corresponding fragments are amplified using detection primers.
  • the primer information is shown in Table 3.
  • the amplified products are sequenced by the Sanger method, and the editing efficiency is determined based on the sequencing results.
  • Example 4 Perform saturation mutation on position 100 of wild-type Cas12j19 and verify its editing activity
  • Example 2 The method of verifying the editing activities of different Cas proteins in animal cells in Example 2 was used to verify the activity of each of the above Cas protein variants in animal cells. The results are shown in Figure 3.
  • a saturation mutation was performed on the 100th amino acid position of the wild-type Cas protein. Different types of amino acids after mutation have different editing activities in animal cells. The 100th position was mutated to K, Y, M. When F, W, S, H, V, I, N, the editing efficiency of the Cas protein variant is significantly higher than that of wild-type Cas12j19.
  • the amino acid mutation is C or L, the activity is basically the same as that of wild-type Cas12j19.
  • the amino acid mutation is Q, The editing activity after G, T, D, and P is lower than that of wild-type Cas12j19.
  • the amino acid type is mutated to A, the Cas protein variant loses its editing activity.

Abstract

The present invention belongs to the field of nucleic acid editing, and particularly relates to the technical field of clustered regularly interspaced short palindromic repeats (CRISPRs). Specifically, the present invention provides a mutated Cas12j protein and use thereof, and the mutated Cas12j protein has wide application prospects.

Description

突变的Cas12j蛋白及其应用Mutated Cas12j protein and its applications
相关申请的交叉引用Cross-references to related applications
本申请要求享有于2022年8月22日提交的名称为“突变的Cas12j蛋白及其应用”的中国专利申请202211005384.7的优先权，上述申请的全部内容通过引用并入本文中。This application claims priority to Chinese patent application 202211005384.7 titled "Mutated Cas12j protein and its application" submitted on August 22, 2022. The entire content of the above application is incorporated herein by reference.
技术领域Technical field
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，本发明涉及一种活性提高的突变的Cas12j蛋白及其应用。The present invention relates to the field of gene editing, in particular to the field of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology. Specifically, the present invention relates to a mutated Cas12j protein with increased activity and its application.
背景技术Background technique
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/Cas technology is a widely used gene editing technology. It uses RNA guidance to specifically bind to target sequences on the genome and cut DNA to produce double-stranded breaks. It uses biological non-homologous end joining or homologous recombination for site-specific Gene editing.
CRISPR/Cas9***是最常用的II型CRISPR***，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***是一类新发现的CRISPR***，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***容易被预测从而降低了潜在的脱靶效应。The CRISPR/Cas9 system is the most commonly used type II CRISPR system. It recognizes the PAM motif of 3’-NGG and performs blunt-end cleavage of the target sequence. The CRISPR/Cas Type V system is a newly discovered CRISPR system that has a 5’-TTN motif and performs sticky end cleavage of target sequences, such as Cpf1, C2c1, CasX, and CasY. However, the different CRISPR/Cas currently existing have different advantages and disadvantages. For example, Cas9, C2c1 and CasX all require two RNAs for guide RNA, while Cpf1 only requires one guide RNA and can be used for multiple gene editing. CasX has a size of 980 amino acids, while the common Cas9, C2c1, CasY and Cpf1 are usually around 1300 amino acids in size. In addition, the PAM sequences of Cas9, Cpf1, CasX, and CasY are relatively 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.
中国发明专利CN111770992B中公开了一种Cas蛋白Cas12j.19，还公开了该蛋白可以在真核细胞中进行基因编辑，但是，其编辑活性并不高，为了提高该蛋白的编辑效率，本申请对该蛋白进行了优化，提高了其在真核细胞中的编辑效率。Chinese invention patent CN111770992B discloses a Cas protein, Cas12j.19, and also discloses that this protein can perform gene editing in eukaryotic cells. However, its editing activity is not high. In order to improve the editing efficiency of this protein, this application The protein was optimized to improve its editing efficiency in eukaryotic cells.
发明内容Contents of the invention
本申请的发明人经过大量实验和反复摸索，通过对Cas12j.19(本申请中将其称之为，Cas12j19)蛋白的定点突变，提高了其编辑活性，扩展了其应用范围。After extensive experiments and repeated explorations, the inventors of the present application improved its editing activity and expanded its application scope through site-directed mutation of the Cas12j.19 (referred to as Cas12j19 in this application) protein.
Cas效应蛋白Cas effector protein
一方面，本发明提供了一种活性改善或活性提高的Cas突变蛋白，所述突变蛋白与亲本Cas蛋白的氨基酸序列相比，在对应于SEQ ID No.1所示氨基酸序列的以下氨基酸位点 处存在突变：第100位。On the one hand, the present invention provides a Cas mutant protein with improved activity or increased activity. Compared with the amino acid sequence of the parent Cas protein, the mutant protein has at the following amino acid positions corresponding to the amino acid sequence shown in SEQ ID No. 1 There is a mutation at position 100.
在一个实施方式中，所述第100位氨基酸突变为非E的氨基酸，例如，A，V，G，L，D，F，W，Y，N，S，Q，K，M，T，C，P，H，R，I；优选，突变为K，Y，M，F，R，W，S，H，V，I，N，C或L。In one embodiment, the amino acid at position 100 is mutated to an amino acid other than E, for example, A, V, G, L, D, F, W, Y, N, S, Q, K, M, T, C , P, H, R, I; preferably, the mutation is K, Y, M, F, R, W, S, H, V, I, N, C or L.
在一个实施方式中，所述亲本Cas蛋白的氨基酸序列与SEQ ID No.1相比具有至少70％、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％、至少99％、至少99.1％、至少99.2％、至少99.3％、至少99.4％、至少99.5％、至少99.6％、至少99.7％、至少99.8％、或至少99.9％的序列同一性。In one embodiment, the amino acid sequence of the parent Cas protein has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% compared to SEQ ID No. 1 , at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity.
在一个实施方式中，所述亲本Cas蛋白为Cas12家族的Cas蛋白，优选，Cas12j家族的Cas蛋白，例如，CN111770992B公开的Cas12j.3-Cas12j.22等；在优选的实施方式中，所述亲本Cas蛋白为Cas12j19；所述Cas12j19的氨基酸序列与SEQ ID No.1相比具有至少70％、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％、至少99％、至少99.1％、至少99.2％、至少99.3％、至少99.4％、至少99.5％、至少99.6％、至少99.7％、至少99.8％、或至少99.9％或100％的序列同一性。In one embodiment, the parent Cas protein is a Cas protein of the Cas12 family, preferably a Cas protein of the Cas12j family, for example, Cas12j.3-Cas12j.22 disclosed in CN111770992B, etc.; in a preferred embodiment, the parent Cas protein Cas protein is Cas12j19; the amino acid sequence of Cas12j19 has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% compared with SEQ ID No.1 %, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, At least 99.8%, or at least 99.9%, or 100% sequence identity.
在一个实施方式中，所述Cas突变蛋白选自以下I-III任意一组：In one embodiment, the Cas mutant protein is selected from any group of I-III below:
I、由SEQ ID No.1所示氨基酸序列在包含以下氨基酸位点处产生突变得到的Cas突变蛋白：第100位；优选的，所述第100位氨基酸突变为非E的氨基酸，例如，A，V，G，L，D，F，W，Y，N，S，Q，K，M，T，C，P，H，R，I；优选，突变为K，Y，M，F，R，W，S，H，V，I，N，C或L；1. Cas mutant protein obtained by mutating the amino acid sequence shown in SEQ ID No. 1 at the following amino acid positions: position 100; preferably, the amino acid at position 100 is mutated to a non-E amino acid, for example, A , V, G, L, D, F, W, Y, N, S, Q, K, M, T, C, P, H, R, I; preferably, the mutation is K, Y, M, F, R , W, S, H, V, I, N, C or L;
II、与I所述的Cas突变蛋白相比，具有I中所述的突变位点；并且，与I所述的Cas突变蛋白相比，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％、至少99％、至少99.1％、至少99.2％、至少99.3％、至少99.4％、至少99.5％、至少99.6％、至少99.7％、至少99.8％、或至少99.9％的序列同一性的Cas突变蛋白；II. Compared with the Cas mutant protein described in I, it has the mutation site described in I; and, compared with the Cas mutant protein described in I, it has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4% , a Cas mutant protein with at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity;
III、与I所述的Cas突变蛋白相比，具有I中所述的突变位点；并且，与I所述的Cas突变蛋白相比，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。III. Compared with the Cas mutant protein described in I, it has the mutation site described in I; and, compared with the Cas mutant protein described in I, it has a sequence with one or more amino acid substitutions, deletions or additions. ; The one or more amino acids include substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids.
来自多种生物体的Cas蛋白或Cas12j蛋白都可以用作亲本Cas蛋白，在一些实施方式中，所述亲本Cas蛋白或Cas12j蛋白具有核酸酶活性。在一些实施方案中，所述亲本Cas蛋白是核酸酶，即切割靶双螺旋核酸(例如，双螺旋DNA)的两条链。在一些实施方案中，所述亲本Cas蛋白是切口酶，即切割靶双螺旋核酸(例如，双螺旋DNA)的单链。Cas proteins or Cas12j proteins from various organisms can be used as parent Cas proteins, and in some embodiments, the parent Cas proteins or Cas12j proteins have nuclease activity. In some embodiments, the parent Cas protein is a nuclease that cleaves both strands of a target duplex nucleic acid (eg, duplex DNA). In some embodiments, the parent Cas protein is a nickase, i.e., cleaves a single strand of a target duplex nucleic acid (eg, duplex DNA).
在一些实施方案中，所述亲本Cas蛋白为天然野生型Cas蛋白；在其他的实施方式中，所述亲本Cas蛋白为经过工程化改造后的Cas蛋白。In some embodiments, the parent Cas protein is a natural wild-type Cas protein; in other embodiments, the parent Cas protein is an engineered Cas protein.
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。It is clear to those skilled in the art that the structure of a protein can be changed without adversely affecting its activity and functionality. For example, one or more conservative amino acid substitutions can be introduced in the amino acid sequence of the protein without affecting the activity and/or functionality of the protein molecule. Or adversely affect the three-dimensional structure. Examples and implementations of conservative amino acid substitutions will be apparent to those skilled in the art. Specifically, the amino acid residue can be replaced with another amino acid residue belonging to the same group as the site to be replaced, that is, a non-polar amino acid residue can be substituted for another non-polar amino acid residue, and a polar uncharged amino acid residue can be substituted. An amino acid residue is substituted for another polar uncharged amino acid residue, a basic amino acid residue is substituted for another basic amino acid residue, and an acidic amino acid residue is substituted for another acidic amino acid residue. Such substituted amino acid residues may or may not be composed of Encoded by the genetic code. Conservative substitutions in which one amino acid is replaced by another amino acid belonging to the same group fall within the scope of the invention as long as the substitution does not result in inactivation of the biological activity of the protein. Therefore, the protein of the present invention may contain one or more conservative substitutions in the amino acid sequence, and these conservative substitutions are preferably produced by substitutions according to Table 1. In addition, the invention also encompasses proteins that also contain one or more other non-conservative substitutions, as long as the non-conservative substitutions do not significantly affect the desired function and biological activity of the protein of the invention.
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。氨基酸置换可以在上述Cas突变蛋白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。Conservative amino acid substitutions can be made at one or more predicted non-essential amino acid residues. "Non-essential" amino acid residues are amino acid residues that can be altered (deletion, substitution or replacement) without altering biological activity, whereas "essential" amino acid residues are required for biological activity. A "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Amino acid substitutions can be made in non-conserved regions of the above-mentioned Cas mutant proteins. 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 fewer conservative or non-conservative changes in conserved regions.
表1
Table 1
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***)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。It is well known in the art that one or more amino acid residues can be altered (substituted, deleted, truncated or inserted) from the N and/or C terminus of a protein while still retaining its functional activity. Therefore, proteins that change one or more amino acid residues from the N and/or C terminus of the 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, which include PCR amplification that alters or extends the protein coding sequence by including the amino acid coding sequence among the oligonucleotides used in the PCR amplification.
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***，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的突变来制备上述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***。It will be appreciated that proteins can be altered in a variety of ways, including amino acid substitutions, deletions, truncation and insertions, and methods for such manipulations are generally known in the art. For example, amino acid sequence variants of the above-mentioned proteins can be prepared by mutating DNA. This can also be accomplished by other forms of mutagenesis and/or by directed evolution, for example using known mutagenesis, recombination and/or shuffling methods in combination with relevant screening methods for single or multiple amino acids. Acid substitutions, deletions and/or insertions.
领域技术人员能够理解，本发明Cas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。Those skilled in the art will understand that these minor amino acid changes in the Cas proteins of the invention can occur (eg, naturally occurring mutations) or be 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 domains of the protein, the properties of the polypeptide may be altered, but the polypeptide may maintain its activity. If mutations are present that are not close to the catalytic domain, active site, or other functional domains, smaller effects can be expected.
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，来鉴定本发明Cas突变蛋白的必需氨基酸。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。Those skilled in the art can identify the essential amino acids of the Cas mutant protein of the present invention according to methods known in the art, such as site-directed mutagenesis or analysis of protein evolution or bioinformatics systems. The catalytic domain, active site or other functional domains of a protein can also be determined by physical analysis of the structure, such as through techniques such as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, combined with putative key sites point amino acid mutations.
本发明中，氨基酸残基可以用单字母表示，也可以用三字母表示，例如：丙氨酸(Ala，A)，缬氨酸(Val，V)，甘氨酸(Gly，G)，亮氨酸(Leu，L)，谷酰胺酸(Gln，Q)，苯丙氨酸(Phe，F)，色氨酸(Trp，W)，酪氨酸(Tyr，Y)，天冬氨酸(Asp，D)，天冬酰胺(Asn，N)，谷氨酸(Glu，E)，赖氨酸(Lys，K)，甲硫氨酸(Met，M)，丝氨酸(Ser，S)，苏氨酸(Thr，T)，半胱氨酸(Cys，C)，脯氨酸(Pro，P)，异亮氨酸(Ile，I)，组氨酸(His，H)，精氨酸(Arg，R)。In the present invention, amino acid residues can be represented by single letters or three letters, for example: alanine (Ala, A), valine (Val, V), glycine (Gly, G), leucine (Leu, L), glutamic acid (Gln, Q), phenylalanine (Phe, F), tryptophan (Trp, W), tyrosine (Tyr, Y), aspartic acid (Asp, D), asparagine (Asn, N), glutamic acid (Glu, E), lysine (Lys, K), methionine (Met, M), serine (Ser, S), threonine (Thr, T), cysteine (Cys, C), proline (Pro, P), isoleucine (Ile, I), histidine (His, H), arginine (Arg, R).
术语“AxxB”表示第xx位的氨基酸A变为氨基酸B，如无特别说明，均是从N端起第xx位的氨基酸A变为氨基酸B。例如，E100R表示第100位的E突变为R。多个氨基酸位点同时存在突变时，可以采用E328R-N369R类似的形式进行表述，例如，E328R-N369R代表第328位E突变为R同时第369位N突变为R。The term "AxxB" means that amino acid A at position xx is changed to amino acid B. Unless otherwise specified, amino acid A at position xx from the N-terminus is changed to amino acid B. For example, E100R means that the E at position 100 is mutated into an R. When mutations exist at multiple amino acid sites at the same time, they can be expressed in a similar form to E328R-N369R. For example, E328R-N369R represents the mutation of E to R at position 328 and N to R at position 369.
本发明所述蛋白质内的特定氨基酸位置(编号)是利用标准序列比对工具通过将目标蛋白质的氨基酸序列与SEQ ID No.1进行比对而确定的，譬如用Smith-Waterman运算法则或用CLUSTALW2运算法则比对两个序列，其中当比对得分最高时认为所述序列是对准的。比对得分可依照Wilbur,W.J.and Lipman,D.J.(1983)Rapid similarity searches ofnucleic acid and protein data banks.Proc.Natl.Acad.Sci.USA,80:726-730中所述的方法进行计算。在ClustalW2(1.82)运算法则中优选使用默认参数：蛋白质缺口开放罚分＝10.0；蛋白质缺口延伸罚分＝0.2；蛋白质矩阵＝Gonnet；蛋白质/DNA端隙＝-1；蛋白质/DNAGAPDIST＝4。优选采用AlignX程序(vectorNTI组中的一部分)，以适于多重比对的默认参数(缺口开放罚分:10og缺口延伸罚分0.05)通过将蛋白质的氨基酸序列与SEQ ID No.1进行比来确定本发明所述蛋白质内特定氨基酸的位置。The specific amino acid position (numbering) within the protein of the present invention is determined by comparing the amino acid sequence of the target protein with SEQ ID No. 1 using standard sequence alignment tools, such as using the Smith-Waterman algorithm or using CLUSTALW2 The algorithm aligns two sequences, with the sequences considered to be aligned when the alignment score is highest. Alignment scores can be calculated according to the method described in Wilbur, W.J. and Lipman, D.J. (1983) Rapid similarity searches of nucleic acid and protein data banks. Proc. Natl. Acad. Sci. USA, 80: 726-730. It is preferred to use the default parameters in the ClustalW2 (1.82) algorithm: protein gap opening penalty=10.0; protein gap extension penalty=0.2; protein matrix=Gonnet; protein/DNA end gap=-1; protein/DNAGAPDIST=4. It is preferably determined by aligning the amino acid sequence of the protein to SEQ ID No. 1 using the AlignX program (part of the vectorNTI group) with default parameters suitable for multiple alignments (gap opening penalty: 10og gap extension penalty 0.05) The position of specific amino acids within the protein of the invention.
在一个实施方式中，所述亲本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％、至少99％、至少99.1％、至少99.2％、至少99.3％、至少99.4％、至少99.5％、至少99.6％、至少99.7％、至少99.8％、或至少99.9％的序列同一性。In one embodiment, the amino acid sequence of the parent Cas protein has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% compared to SEQ ID No. 1 , at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity.
在一个实施方式中，所述Cas突变蛋白选自以下I-III任意一组：In one embodiment, the Cas mutant protein is selected from any group of I-III below:
I、由SEQ ID No.1所示氨基酸序列在包含以下任一或任意几个氨基酸位点处产生突变得到的Cas突变蛋白：第100位；I. A Cas mutant protein obtained by mutating the amino acid sequence shown in SEQ ID No. 1 at any one or several of the following amino acid positions: position 100;
II、与I所述的Cas突变蛋白相比，具有I中所述的突变位点；并且，与I所述的Cas突变蛋白相比，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％的序列同一性的Cas突变蛋白；II. Compared with the Cas mutant protein described in I, it has the mutation site described in I; and, compared with the Cas mutant protein described in I, it has at least 80%, at least 85%, at least 90%, at least A Cas mutant protein with 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity;
III、与I所述的Cas突变蛋白相比，具有I中所述的突变位点；并且，与I所述的Cas突变蛋白相比，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。III. Compared with the Cas mutant protein described in I, it has the mutation site described in I; and, compared with the Cas mutant protein described in I, it has a sequence with one or more amino acid substitutions, deletions or additions. ; the one or more Amino acids include substitutions, deletions, or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.
所述Cas蛋白的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。The biological functions of the Cas protein include, but are not limited to, the activity of binding to guide RNA, endonuclease activity, and the activity of binding to and cutting specific sites in the target sequence under the guidance of guide RNA, including but not limited to Cis cleavage activity. and Trans cleavage activity.
本发明中，“Cas突变蛋白”也可以称之为突变的Cas蛋白，或者Cas蛋白变体。In the present invention, "Cas mutant protein" can also be called a mutated Cas protein or a Cas protein variant.
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突变蛋白和其他的修饰部分。The present invention also provides a fusion protein, which includes the above-mentioned Cas mutant protein and other modified parts.
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。In one embodiment, the modifying moiety is selected from another protein or polypeptide, a detectable label, or any combination thereof.
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，腺苷脱氨酶，胞苷脱氨酶，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。In one embodiment, the modified moiety is selected from an epitope tag, a reporter gene sequence, a nuclear localization signal (NLS) sequence, a targeting moiety, a transcriptional activation domain (e.g., VP64), a transcriptional repression domain (e.g., KRAB domain or SID domain), a nuclease domain (e.g., Fok1), and a domain with an activity selected from: nucleotide deaminase, adenosine deaminase, cytidine deaminase, methyl enzyme activity, demethylase, transcription activation activity, transcription repression 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 any combination thereof. The NLS sequence is well known to those skilled in the art, and examples thereof include, but are not limited to, SV40 large T antigen, EGL-13, c-Myc and TUS protein.
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)。In one embodiment, the NLS sequence is located at, near or close to the end (eg, N-terminus, C-terminus or both ends) of the Cas protein of the invention.
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)。The 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 Tags (e.g., for purification, detection, or tracing).
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。The reporter gene sequence is well known to those skilled in the art, and examples thereof include but are not limited to GST, HRP, CAT, GFP, HcRed, DsRed, CFP, YFP, BFP, etc.
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。In one embodiment, the fusion protein of the present invention includes 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, etc.
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。In one embodiment, the fusion proteins of the invention comprise a detectable label, such as a fluorescent dye, such as FITC or 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。In one embodiment, the Cas protein of the invention is coupled, conjugated or fused to the modified moiety, optionally via a linker.
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。In one embodiment, the modified moiety is directly linked to the N-terminus or C-terminus of the Cas protein of the invention.
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。In one embodiment, the modified portion is connected to the N-terminus or C-terminus of the Cas protein of the invention through a linker. Such linkers are well known in the art and examples include, but are not limited to, those containing one or more (eg, 1, 2, 3, 4 or 5) amino acids (eg, Glu or Ser) or amino acid derivatives (such as Ahx, β-Ala, GABA or Ava) linkers, or PEG, etc.
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。The Cas protein, protein derivative or fusion protein of the present invention is not limited by its production method. For example, it can be produced by genetic engineering methods (recombinant technology) or chemical synthesis methods.
Cas蛋白的核酸Cas protein nucleic acid
另一方面，本发明提供了一种分离的多核苷酸，其包含：In another aspect, the invention provides an isolated polynucleotide comprising:
(a)编码本发明的Cas突变蛋白或融合蛋白的多核苷酸序列；(a) The polynucleotide sequence encoding the Cas mutant protein or fusion protein of the present invention;
或者，与(a)所述的多核苷酸互补的多核苷酸。Or, a polynucleotide complementary to the polynucleotide described in (a).
在一个实施方式中，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，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。In one embodiment, the nucleotide sequence is codon-optimized for expression in prokaryotic cells. exist In one embodiment, the nucleotide sequence is codon-optimized for expression in eukaryotic cells.
在一个实施方式中，所述细胞是动物细胞，例如，哺乳动物细胞。In one embodiment, the cells are animal cells, eg, mammalian cells.
在一个实施方式中，所述细胞是人类细胞。In one embodiment, the cells are human cells.
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。In one embodiment, the cells are plant cells, for example cells of cultivated plants (such as cassava, corn, sorghum, wheat or rice), algae, trees or vegetables.
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。In one embodiment, the polynucleotide is preferably single-stranded or double-stranded.
指导RNA(gRNA)Guide 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”。On the other hand, the present invention provides a gRNA, which includes a first segment and a second segment; the first segment is also called a “skeleton region”, “protein binding segment”, “protein binding segment” Sequence", or "Direct Repeat (Direct Repeat) sequence"; the second segment is also called "targeting sequence for targeting nucleic acid" or "targeting segment for targeting nucleic acid", or "targeting target" sequence's boot sequence".
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。The first segment of the gRNA can interact with the Cas protein of the present invention, so that the Cas protein and the gRNA form a complex.
在优选的实施方式中，所述第一区段为如上所述的同向重复序列。In a preferred embodiment, the first segment is a direct repeat sequence as described above.
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。The targeting sequence of the targeting nucleic acid or the targeting segment of the targeting nucleic acid of the invention comprises a nucleotide sequence complementary to a sequence in the target nucleic acid. In other words, the targeting sequence or the targeting segment of the targeting nucleic acid of the invention interacts with the target nucleic acid in a sequence-specific manner through hybridization (ie, base pairing). Thus, the targeting sequence of a targeting nucleic acid or the targeting segment of a targeting nucleic acid may be altered, or may be modified to hybridize to any desired sequence within the target nucleic acid. The nucleic acid is selected from DNA or RNA.
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％)。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% (e.g., at least 65%, at least 70%, at least 75%, at least 80% , at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100%).
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。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 Cas protein). The gRNA of the present invention guides its interacting Cas protein to the specific nucleotide sequence within the target nucleic acid through the action of the targeting sequence of the targeted nucleic acid.
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。Preferably, the guide RNA includes a first segment and a second segment from the 5' to 3' direction.
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。In the present invention, the second segment can also be understood as a guide sequence that hybridizes to the target sequence.
本发明的gRNA能够与所述Cas蛋白形成复合物。The gRNA of the present invention can form a complex with the Cas protein.
载体carrier
本发明还提供了一种载体，其包含如上述的Cas突变蛋白、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。The present invention also provides a vector, which includes the above-mentioned Cas mutant protein, isolated nucleic acid molecule or polynucleotide; preferably, it also includes a regulatory element operably connected thereto.
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。In one embodiment, the regulatory element is selected from one or more of the following group: enhancer, transposon, promoter, terminator, leader sequence, polyadenylation sequence, and marker gene.
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。In one embodiment, the vector includes a cloning vector, an expression vector, a shuttle vector, and an integration vector.
在一些实施方案中，所述***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。In some embodiments, the vectors included in the system are viral vectors (e.g., retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated vectors, and herpes simplex vectors), and may also be plasmids, viruses, cosmids, Phage and other types, which are well known to those skilled in the art.
CRISPR***CRISPR system
本发明提供了一种工程化的非天然存在的载体***，或者是CRISPR-Cas***，该***包括Cas突变蛋白或编码所述Cas突变蛋白的核酸序列以及编码一种或多种指导RNA的核酸。The invention provides an engineered non-naturally occurring vector system, or a CRISPR-Cas system, which system includes a Cas mutant protein or a nucleic acid sequence encoding the Cas mutant protein and a nucleic acid encoding one or more guide RNAs. .
在一种实施方式中，所述编码所述Cas突变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。In one embodiment, the nucleic acid sequence encoding the Cas mutant protein and the nucleic acid encoding one or more guide RNAs are artificially synthesized.
在一种实施方式中，所述编码所述Cas突变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。In one embodiment, the nucleic acid sequence encoding the Cas mutant protein and encoding one or more guides The nucleic acid RNA does not occur naturally together.
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。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 guide the Cas protein to the genomic locus of the DNA molecule of the one or more gene products. The Cas protein Upon reaching the target sequence location, the target sequence is modified, edited, or cleaved, whereby the expression of the one or more gene products is altered or modified.
本发明的细胞包括动物、植物或微生物中的一种或多种。The cells of the present invention include one or more of animals, plants or microorganisms.
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。In some embodiments, the Cas protein is codon-optimized for expression in cells.
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。In some embodiments, the Cas protein directs cleavage of one or both strands at the target sequence location.
本发明还提供了一种工程化的非天然存在的载体***，该载体***可以包括一种或多种载体，该一种或多种载体包括：The present invention also provides an engineered non-naturally occurring vector system. The vector system may include one or more vectors, and the one or more vectors include:
a)第一调控元件，该第一调控元件可操作地与gRNA连接，a) a first regulatory element operably linked to the gRNA,
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；b) a second regulatory element operably connected to the Cas protein;
其中组分(a)和(b)位于该***的相同或不同载体上。wherein components (a) and (b) are located on the same or different carriers of the system.
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)。The first and second regulatory elements include promoters (e.g., constitutive promoters or inducible promoters), enhancers (e.g., 35S promoter or 35S enhanced promoter), internal ribosome entry sites (IRES), and others. Expression control elements (eg, transcription termination signals, such as polyadenylation signals and polyU sequences).
在一些实施方案中，所述***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。In some embodiments, the vector in the system is a viral vector (eg, retroviral vector, lentiviral vector, adenoviral vector, adeno-associated vector, and herpes simplex vector), and may also be a plasmid, virus, cosmid, phage and other types, which are well known to those skilled in the art.
在一些实施例中，本文提供的***处于递送***中。在一些实施方案中，递送***是纳米颗粒，脂质体，外体，微泡和基因枪。In some embodiments, the systems provided herein are in a delivery system. In some embodiments, delivery systems are nanoparticles, liposomes, exosomes, microbubbles, and gene guns.
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。In one embodiment, the target sequence is a DNA or RNA sequence from a prokaryotic or eukaryotic cell. In one embodiment, the target sequence is a non-naturally occurring DNA or RNA sequence.
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。In one embodiment, the target sequence is present within the cell. In one embodiment, the target sequence is present in the nucleus or cytoplasm (eg, organelle). In one embodiment, the cell is a eukaryotic cell. In other embodiments, the cell is a prokaryotic cell.
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。In one embodiment, the Cas protein is linked to one or more NLS sequences. In one embodiment, the fusion protein contains one or more NLS sequences. In one embodiment, the NLS sequence is linked to the N-terminus or C-terminus of the protein. In one embodiment, the NLS sequence is fused to the N-terminus or C-terminus of the protein.
另一方面，本发明涉及一种工程化的CRISPR***，所述***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。In another aspect, 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 include direct repeats and spacers capable of hybridizing to target nucleic acids. sequence, the Cas protein is capable of binding the guide RNA and targeting a target nucleic acid sequence complementary to the spacer sequence.
蛋白-核酸复合物/组合物Protein-nucleic acid complexes/compositions
另一方面，本发明提供了一种复合物或者组合物，其包含：In another aspect, the invention provides a complex or composition comprising:
(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(i) Protein component, which is selected from: the above-mentioned Cas protein, derivatized protein or fusion protein, and any combination thereof; and
(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。(ii) A nucleic acid component 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 invention.
所述蛋白组分与核酸组分相互结合形成复合物。The protein component and the nucleic acid component combine with each other to form a complex.
在一个实施方式中，所述核酸组分是CRISPR-Cas***中的指导RNA。In one embodiment, the nucleic acid component is a guide RNA in a CRISPR-Cas system.
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。 In one embodiment, the complex or composition is non-naturally occurring or modified. In one embodiment, at least one component of the complex or composition is non-naturally occurring or modified. In one embodiment, the first component is non-naturally occurring or modified; and/or the second component is non-naturally occurring or modified.
活化的CRISPR复合物Activated CRISPR complex
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。On the other hand, the present invention also provides an activated CRISPR complex, which includes: (1) a protein component selected from: the Cas protein, derivatized protein or fusion protein of the present invention, and any combination thereof; (2) gRNA, which includes (a) a guide sequence capable of hybridizing to the target sequence; and (b) a direct repeat sequence capable of binding to the Cas protein of the invention; and (3) binding to the gRNA target sequence. Preferably, the binding is binding to the target nucleic acid through a targeting sequence on the gRNA that targets the nucleic acid.
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***中Cas蛋白、gRNA与靶核酸结合或修饰后的复合物。The terms "activated CRISPR complex", "activated complex" or "ternary complex" used herein refer to the complex in which Cas protein, gRNA and target nucleic acid are combined or modified in the CRISPR system.
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。The Cas protein and gRNA of the present invention can form a binary complex, which is activated when combined with a nucleic acid substrate to form an activated CRISPR complex. The nucleic acid substrate and the spacer sequence (or so-called spacer sequence) in the gRNA are , complementary to the guide sequence that hybridizes to the target nucleic acid). In some embodiments, the gRNA's spacer sequence exactly matches the target substrate. In other embodiments, the spacer sequence of the gRNA matches a portion (contiguous or discontinuous) of the target substrate.
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。In a preferred embodiment, the activated CRISPR complex can exhibit side-branching nucleic acid cleavage activity, and the side-branching nucleic acid cleavage activity refers to the non-specific cleavage activity or random cleavage activity of single-stranded nucleic acids exhibited by the activated CRISPR complex. Cleavage activity is also called trans cleavage activity in the art.
递送及递送组合物Delivery and delivery compositions
本发明的Cas蛋白、gRNA、融合蛋白、核酸分子、载体、***、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。The Cas protein, gRNA, fusion protein, nucleic acid molecule, vector, system, complex and composition 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, dendrimers Transfection, heat shock transfection, nucleofection, magnetofection, lipofection, puncture transfection, optical transfection, reagent-enhanced nucleic acid uptake, and via liposomes, immunoliposomes, virus particles, artificial viruses Delivery of body etc.
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、***、复合物和组合物。Therefore, in another aspect, the present invention provides a delivery composition, which includes a delivery vector, and one or any several selected from the following: Cas protein, fusion protein, nucleic acid molecule, vector, system, Complexes and compositions.
在一个实施方式中，所述递送载体是粒子。In one embodiment, the delivery vehicle is a particle.
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)。In one embodiment, the delivery vehicle is selected from lipid particles, sugar particles, metal particles, protein particles, liposomes, exosomes, microvesicles, gene guns or viral vectors (e.g., replication-deficient retroviruses , lentivirus, adenovirus or adeno-associated virus).
宿主细胞host cell
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。The present invention also relates to an in vitro, ex vivo or in vivo cell or cell line or their progeny, which cell or cell line or their progeny contains: Cas protein, fusion protein, nucleic acid according to the invention Molecules, protein-nucleic acid complexes, activated CRISPR complexes, vectors, delivery compositions of the invention.
在某些实施方案中，所述细胞是原核细胞。In certain embodiments, the cell is a prokaryotic cell.
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)。In certain embodiments, the cell is a eukaryotic cell. In certain embodiments, the cells are mammalian cells. In certain embodiments, the cells are human cells. In certain embodiments, the cell is a non-human mammalian cell, such as a non-human primate, bovine, ovine, porcine, canine, monkey, rabbit, rodent (eg, rat or mouse) cell. In certain embodiments, the cell is a non-mammalian eukaryotic cell, such as a cell of a poultry bird (eg, chicken), fish, or crustacean (eg, clam, shrimp). In certain embodiments, the cells are plant cells, such as those of a monocot or dicot or a cultivated plant or a food crop such as cassava, corn, sorghum, soybean, wheat, oat or rice, e.g. Algae, trees or production plants, fruits or vegetables (for example, trees such as citrus trees, nut trees; nightshades, cotton, tobacco, tomatoes, grapes, coffee, cocoa, etc.).
在某些实施方案中，所述细胞是干细胞或干细胞系。In certain embodiments, the cells are stem cells or stem cell lines.
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。 In some cases, a host cell of the invention contains a genetic or genomic modification that is not present in its wild type.
基因编辑方法和应用Gene editing methods and applications
本发明的Cas突变蛋白、核酸、上述组合物、上述CIRSPR/Cas***、上述载体***、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。The Cas mutant protein, nucleic acid, the above composition, the above CIRSPR/Cas system, the above vector system, the above delivery composition or the above activated CRISPR complex or the above host cell of the present invention can be used for any one or any several of the following purposes: target To and/or edit target nucleic acid; cleave double-stranded DNA, single-stranded DNA or single-stranded RNA; non-specific cleavage and/or degradation of side branch nucleic acids; non-specific cleavage of single-stranded nucleic acids; nucleic acid detection; detection of nucleic acids in target samples; specificity Base editing of double-stranded nucleic acids; base editing of double-stranded nucleic acids; base editing of single-stranded nucleic acids. In other embodiments, it can also be used to prepare reagents or kits for any one or several of the above uses.
本发明还提供了上述Cas蛋白、核酸、上述组合物、上述CIRSPR/Cas***、上述载体***、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。The invention also provides the application of the above-mentioned Cas protein, nucleic acid, above-mentioned composition, above-mentioned CIRSPR/Cas system, above-mentioned vector system, above-mentioned delivery composition or above-mentioned activated CRISPR complex in gene editing, gene targeting or gene cutting; Alternatively, use in the preparation of reagents or kits for gene editing, gene targeting or gene cleavage.
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。In one embodiment, the gene editing, gene targeting or gene cleavage is performed intracellularly and/or extracellularly.
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***、上述载体***、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。The invention also provides a method for editing target nucleic acid, targeting target nucleic acid or cutting target nucleic acid. The method includes combining the target nucleic acid with the above-mentioned Cas protein, nucleic acid, above-mentioned composition, above-mentioned CIRSPR/Cas system, above-mentioned vector system, The above delivery composition or the above activated CRISPR complex is contacted. In one embodiment, the method is editing, targeting, or cleaving a target nucleic acid intracellularly or extracellularly.
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***多核苷酸、基因突变。The gene editing or editing target nucleic acid includes modifying genes, knocking out genes, changing the expression of gene products, repairing mutations, and/or inserting polynucleotides, and gene mutations.
所述编辑可以在原核细胞和/或真核细胞中进行编辑。The editing can be performed in prokaryotic cells and/or eukaryotic cells.
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***、上述载体***、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。On the other hand, 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 nucleic acid detection, or in the preparation of Use in reagents or kits for nucleic acid detection.
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。On the other hand, the present invention also provides a method for cutting single-stranded nucleic acids, which method includes contacting a nucleic acid population with the above-mentioned Cas protein and gRNA, wherein the nucleic acid population includes 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.
所述gRNA能够结合所述Cas蛋白。The gRNA is capable of binding to the Cas protein.
所述gRNA能够靶向所述靶核酸。The gRNA is capable of targeting the target nucleic acid.
所述接触可以是在体外、离体或体内的细胞内部。The contact may be inside the cell in vitro, ex vivo or in vivo.
优选的，所述切割单链核酸为非特异性的切割。Preferably, the cleavage of single-stranded nucleic acid is non-specific cleavage.
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***、上述载体***、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。On the other hand, 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 vector system, the above-mentioned delivery composition or the above-mentioned activated CRISPR complex in non-specific cutting of single-stranded nucleic acids. Application, or use in the preparation of reagents or kits for non-specific cleavage of single-stranded nucleic acids.
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***、上述载体***、上述递送组合物、上述活化的CRISPR复合物或上述宿主细胞。On the other hand, the present invention also provides a kit for gene editing, gene targeting or gene cutting, which kit includes the above-mentioned Cas protein, gRNA, nucleic acid, the above-mentioned composition, the above-mentioned CIRSPR/Cas system, the above-mentioned The vector system, the above-mentioned delivery composition, the above-mentioned activated CRISPR complex or the above-mentioned host cell.
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。On the other hand, the present invention also provides a kit for detecting target nucleic acid in a sample, the kit comprising: (a) Cas protein, or nucleic acid encoding the Cas protein; (b) 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.
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。It is known in the art that precursor RNA can be cleaved or processed into the mature guide RNA described above.
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***、上述载体***、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：On the other hand, the invention provides the above-mentioned Cas protein, nucleic acid, above-mentioned composition, above-mentioned CIRSPR/Cas system, above-mentioned vector system, above-mentioned delivery composition, above-mentioned activated CRISPR complex or above-mentioned host cell in preparation preparations or kits Purpose, the preparation or kit is used for:
(i)基因或基因组编辑； (i) Gene or genome editing;
(ii)靶核酸检测和/或诊断；(ii) Target nucleic acid detection and/or diagnosis;
(iii)编辑靶基因座中的靶序列来修饰生物或非人类生物；(iii) Edit target sequences in target loci to modify organisms or non-human organisms;
(iv)疾病的治疗；(iv) Treatment of disease;
(iv)靶向靶基因。(iv) Targeting target genes.
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。Preferably, the above-mentioned gene or genome editing is performed within or outside the cell.
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。Preferably, the target nucleic acid detection and/or diagnosis is performed in vitro.
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。Preferably, the treatment of the disease is the treatment of a condition caused by a defect in a target sequence in a target locus.
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。In another aspect, the 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 Cas protein-binding region and the guide sequence for hybridizing with the target nucleic acid; detecting the detectable signal generated by the Cas protein cutting single-stranded nucleic acid detector, thereby detecting the target nucleic acid; the single-stranded nucleic acid detector does not hybridize with the gRNA .
特异性修饰靶核酸的方法Methods for specifically modifying target nucleic acids
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***、上述载体***、上述递送组合物或上述活化的CRISPR复合物接触。On the other hand, the present invention also provides a method for specifically modifying a target nucleic acid. The method includes: combining the target nucleic acid with the above-mentioned Cas protein, nucleic acid, above-mentioned composition, above-mentioned CIRSPR/Cas system, above-mentioned carrier system, and above-mentioned delivery composition. or contact with the activated CRISPR complex described above.
该特异性修饰可以发生在体内或者体外。The specific modification can occur in vivo or in vitro.
该特异性修饰可以发生在细胞内或者细胞外。The specific modification can occur intracellularly or extracellularly.
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。In some cases, the cells are selected from prokaryotic or eukaryotic cells, for example, animal cells, plant cells, or microbial cells.
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。In one embodiment, the modification refers to a break in the target sequence, such as a single/double strand break in DNA, or a single strand break in RNA.
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。In some cases, the method further includes 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 Part of the copy is integrated into the target nucleic acid.
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)***所述断裂中。In one embodiment, the modification further includes inserting an editing template (eg, exogenous nucleic acid) into the break.
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***、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。In one embodiment, the method further includes contacting an editing template with the target nucleic acid or delivering it to a cell containing the target nucleic acid. In this embodiment, the method repairs the broken target gene by homologous recombination with an exogenous template polynucleotide; in some embodiments, the repair results in a mutation that includes one or more of the target gene Insertions, deletions, or substitutions of multiple nucleotides, and in other embodiments, the mutations result in one or more amino acid changes in the protein expressed from the gene containing the target sequence.
检测(非特异切割)Detection (non-specific cleavage)
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***、上述载体***、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。On the other hand, the present invention provides a method for detecting target nucleic acid in a sample, the method comprising combining the sample with the above-mentioned Cas protein, nucleic acid, above-mentioned composition, above-mentioned CIRSPR/Cas system, above-mentioned carrier system, above-mentioned delivery composition or The above-mentioned activated CRISPR complex is in contact with the single-stranded nucleic acid detector; the detectable signal generated by the Cas protein cutting the single-stranded nucleic acid detector is detected, thereby detecting the target nucleic acid.
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。In the present invention, the target nucleic acid includes ribonucleotides or deoxyribonucleotides; includes single-stranded nucleic acid and double-stranded nucleic acid, such as single-stranded DNA, double-stranded DNA, single-stranded RNA, and double-stranded 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。In one embodiment, the target nucleic acid is derived from samples such as viruses, bacteria, microorganisms, soil, water sources, human bodies, animals, plants, etc. Preferably, the target nucleic acid is a product enriched or amplified by PCR, NASBA, RPA, SDA, LAMP, HAD, NEAR, MDA, RCA, LCR, RAM and other methods.
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，***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。In one embodiment, the target nucleic acid is a viral nucleic acid, a bacterial nucleic acid, a specific nucleic acid related to a disease, such as a specific mutation site or SNP site or a nucleic acid that is different from a control; preferably, the virus is a plant Viruses or animal viruses, for example, papillomavirus, hepatovirus, herpesvirus, adenovirus, poxvirus, parvovirus, coronavirus; preferably, the virus is a coronavirus, preferably SARS, SARS-CoV2 (COVID-19) -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％。In the present invention, the gRNA has a matching degree of at least 50% 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%.
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。In one embodiment, when the target sequence contains one or more characteristic sites (such as specific mutation sites or SNPs), the characteristic sites completely match the gRNA.
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。In one embodiment, the detection method may include one or more gRNAs with different guide sequences targeting different target sequences.
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、***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。In the present invention, the single-stranded nucleic acid detector includes but is not limited to single-stranded DNA, single-stranded RNA, DNA-RNA hybrids, nucleic acid analogs, base modifications, and single-stranded nucleic acid detection containing abase spacers. devices, etc.; "nucleic acid analogs" include but are not limited to: locked nucleic acid, bridged nucleic acid, morpholine nucleic acid, ethylene 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' sulfur RNA, and combinations thereof, including optional ribonucleotide or deoxyribonucleotide residues.
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。In the present invention, the detectable signal is achieved in the following ways: vision-based detection, sensor-based detection, color detection, fluorescence signal-based detection, gold nanoparticle-based detection, fluorescence polarization, colloidal phase change/dispersion, electrochemical Chemical detection and semiconductor-based detection.
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。In the present invention, preferably, the two ends of the single-stranded nucleic acid detector are respectively provided with fluorescent groups and quenching groups. When the single-stranded nucleic acid detector is cleaved, it can show a detectable fluorescence signal. The fluorescent group is selected from one or more of FAM, FITC, VIC, JOE, TET, CY3, CY5, ROX, Texas Red or LC RED460; the quenching group is selected from BHQ1, BHQ2, BHQ3 , Dabcy1 or Tamra, or any one of them.
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。In other embodiments, the 5' end and 3' end of the single-stranded nucleic acid detector are respectively provided with different labeling molecules, and colloidal gold detection is used to detect the time before and after the single-stranded nucleic acid detector is cleaved by the Cas protein. Colloidal gold test results after being cleaved by Cas protein; the single-stranded nucleic acid detector will show different color results on the colloidal gold detection line and quality control line before being cleaved by Cas protein and after being cleaved by Cas protein.
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。In some embodiments, the method of detecting a target nucleic acid may further include 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.
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。In some embodiments, the method of detecting a target nucleic acid may further include using an RNA reporter nucleic acid and a DNA reporter nucleic acid on different channels (e.g., fluorescent colors), and by measuring 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 is used to determine the level of detectable signal, and the level of detectable signal is sampled based on combining (eg, using a minimum or product).
在一个实施方式中，所述靶基因存在于细胞内。In one embodiment, the target gene is present within the cell.
在一个实施方式中，所述细胞是原核细胞。In one embodiment, the cell is a prokaryotic cell.
在一个实施方式中，所述细胞是真核细胞。In one embodiment, the cell is a eukaryotic cell.
在一个实施方式中，所述细胞是动物细胞。In one embodiment, the cells are animal cells.
在一个实施方式中，所述细胞是人类细胞。In one embodiment, the cells are human cells.
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。In one embodiment, the cells are plant cells, for example cells of cultivated plants (such as cassava, corn, sorghum, wheat or rice), algae, trees or vegetables.
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。In one embodiment, the target gene is present in a nucleic acid molecule (eg, a plasmid) in vitro.
在一个实施方式中，所述靶基因存在于质粒中。In one embodiment, the target gene is present in a plasmid.
术语定义Definition of Terms
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。In the present invention, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Moreover, the procedures used in this article such as molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics and recombinant DNA are routine procedures widely used in the corresponding fields. . Meanwhile, in order to better understand the present invention, definitions of relevant terms and explain.
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。Nucleic acid cleavage or cleavage of nucleic acids herein includes: DNA or RNA cleavage in the target nucleic acid produced by the Cas enzyme described herein (Cis cleavage), DNA or RNA cleavage in side nucleic acid substrates (single-stranded nucleic acid substrates) ( That is, non-specific or non-targeted, Trans cleavage). In some embodiments, the cleavage is a double-stranded DNA break. In some embodiments, the cleavage is a single-stranded DNA break or a single-stranded RNA break.
CRISPR***CRISPR system
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)***”或“CRISPR***”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。As used herein, the term "Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) (CRISPR-Cas) system" or "CRISPR system" is used interchangeably and has the skill in the art. It is generally understood that it generally includes transcripts or other elements related to the expression of CRISPR-related ("Cas") genes, or transcripts or other elements capable of directing the activity of the Cas gene.
CRISPR/Cas复合物CRISPR/Cas complex
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。As used herein, the term "CRISPR/Cas complex" refers to a complex formed by binding of guide RNA (guide RNA) or mature crRNA to Cas protein, which includes a guide sequence that hybridizes to the target sequence and is Protein-bound direct repeats allow the complex to recognize and cleave polynucleotides that hybridize to the guide RNA or mature crRNA.
指导RNA(guideRNA，gRNA)Guide RNA (guideRNA, 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。As used herein, the terms "guide RNA (gRNA)," "mature crRNA," and "guide sequence" are used interchangeably and have meanings commonly understood by those skilled in the art. Generally speaking, a guide RNA may comprise, consist essentially of, or consist of direct repeats and a leader sequence.
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。In some cases, a guide sequence is any polynucleotide sequence that has sufficient complementarity to a target sequence to hybridize to the target sequence and direct specific binding of the CRISPR/Cas complex to the target sequence. In one embodiment, when optimally aligned, the degree of complementarity between 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 public and commercially available alignment algorithms and programs such as, but not limited to, ClustalW, Smith-Waterman in matlab, Bowtie, Geneious, Biopython, and SeqMan.
靶序列target sequence
“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。"Target sequence" refers to a polynucleotide targeted by a guide sequence in a gRNA, e.g., 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) formation. Perfect complementarity is not required, as long as sufficient complementarity exists to cause hybridization and promote the formation of a CRISPR/Cas complex.
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。The target sequence can comprise any polynucleotide, such as DNA or RNA. In some cases, the target sequence is located intracellularly or extracellularly. In some cases, the target sequence is located in the nucleus or cytoplasm of the cell. In some cases, the target sequence may be located in an organelle of a eukaryotic cell such as a mitochondria or chloroplast. The sequence or template that can be used for recombination into a target locus containing the target sequence is called an "editing template" or "editing polynucleotide" or "editing sequence." In one embodiment, the editing template is an exogenous nucleic acid. In one embodiment, the recombination is homologous recombination.
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。In the present invention, a "target sequence" or "target polynucleotide" or "target nucleic acid" may be any polynucleotide endogenous or exogenous to a cell (eg, a eukaryotic cell). For example, the target polynucleotide may 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 useless DNA). In some cases, the target sequence should be associated with a protospacer adjacent motif (PAM).
单链核酸检测器Single-stranded nucleic acid detector
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。The single-stranded nucleic acid detector of the present invention refers to a sequence containing 2-200 nucleotides, preferably, having 2-150 nucleotides. Nucleotides are preferably 3-100 nucleotides, preferably 3-30 nucleotides, preferably 4-20 nucleotides, and more preferably 5-15 nucleotides. Preferred are single-stranded DNA molecules, single-stranded RNA molecules or single-stranded DNA-RNA hybrids.
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。The two ends of the single-stranded nucleic acid detector include different reporting groups or labeling molecules. When it is in the initial state (that is, when it is not cleaved), it does not present a reporting signal. When the single-stranded nucleic acid detector is cleaved, it displays a reporting signal. A detectable signal is produced, that is, there is a detectable difference after cutting and before cutting.
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。In one embodiment, the reporter group or labeling molecule includes a fluorescent group and a quenching group, and the fluorescent group is selected from FAM, FITC, VIC, JOE, TET, CY3, CY5, ROX, Texas Red Or one or any several of LC RED460; the quenching group is selected from one or any of several BHQ1, BHQ2, BHQ3, Dabcy1 or Tamra.
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。In one embodiment, the single-stranded nucleic acid detector has a first molecule (such as FAM or FITC) connected to the 5' end and a second molecule (such as biotin) connected to the 3' end. The reaction system containing a single-stranded nucleic acid detector is used in conjunction with a flow strip to detect target nucleic acids (preferably, colloidal gold detection method). The flow strip is designed to have two capture lines. The sample contact end (colloidal gold) is provided with an antibody that binds the first molecule (i.e., the first molecule antibody), and the first line (control line) contains an antibody that binds the third molecule. An antibody with one molecule of antibody contains a second molecule of antibody (that is, a second molecule of antibody, such as avidin) that binds to the second molecule at the second line (test line). As the reaction flows along the strip, the first molecule of antibody binds to the first molecule and carries the cleaved or uncleaved oligonucleotide to the capture line. The cleaved reporter will bind to the first molecule of antibody at the first capture line. antibody, while the uncleaved reporter will bind a second antibody molecule at the second capture line. The binding of reporter groups in each line will result in a strong readout/signal (eg color). As more reporters are cleaved, more signal will accumulate at the first capture line, and less signal will appear at the second line. In certain aspects, the invention relates to the use of a flow strip as described herein for detecting nucleic acids. In certain aspects, the invention relates to methods of detecting nucleic acids using flow strips as defined herein, such as (lateral) flow tests or (lateral) flow immunochromatographic assays. In some aspects, the molecules in the single-stranded nucleic acid detector can be replaced with each other, or the position of the molecules can be changed. As long as the reporting principle is the same or similar to the present invention, the improved methods are also included in the present invention.
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。The detection method of the present invention can be used for quantitative detection of target nucleic acids to be detected. The quantitative detection index can be quantified based on the signal strength of the reporter group, such as the luminescence intensity of the fluorescent group, or the width of the color band.
野生型Wild type
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。As used herein, the term "wild type" has the meaning commonly understood by those skilled in the art to mean the typical form of an organism, strain, gene, or characteristics that distinguish it from mutant or variant forms as it occurs in nature, It is isolated from sources in nature and has not been intentionally modified by man.
衍生化derivatization
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。As used herein, the term "derivatization" refers to the chemical modification of an amino acid, polypeptide, or protein to which one or more substituents have been covalently linked. Substituents may also be called side chains.
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。A derivatized protein is a derivative of the protein. Generally, derivatization of the protein does not adversely affect the desired activity of the protein (e.g., binding activity to guide RNA, endonuclease activity, binding to a target sequence under the guidance of guide RNA) Specific site binding and cleavage activity), that is to say, the protein derivatives have the same activity as the protein.
衍生化蛋白derivatized protein
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、***、修饰和/或取代。Also known as "protein derivatives", refers to modified forms of proteins, for example, in which one or more amino acids of the protein can be deleted, inserted, modified and/or substituted.
非天然存在的non-naturally occurring
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。As used herein, the terms "non-naturally occurring" or "engineered" are used interchangeably and mean the involvement of man. When these terms are used to describe a nucleic acid molecule or polypeptide, it is meant that the nucleic acid molecule or polypeptide is at least substantially free from at least one other component with which it is associated in nature or as found in nature.
直系同源物(orthologue,ortholog)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。As used herein, the term "orthologue, ortholog" has the meaning commonly understood by those skilled in the art. As further guidance, an "ortholog" of a protein as used herein refers to a protein belonging to a different species that performs the same or similar function as the protein of which it is an ortholog.
同一性Identity
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。As used herein, the term "identity" is used to refer to the match of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (for example, a position in each of two DNA molecules is occupied by adenine, or two A certain position in each polypeptide is occupied by lysine), then the molecules are identical at that position. "Percent identity" between two sequences is a function of the number of matching positions common to the two sequences divided by the number of positions compared × 100. For example, if 6 out of 10 positions of two sequences match, then the two sequences are 60% identical. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (matching at 3 positions out of a total of 6 positions). Typically, comparisons are made when two sequences are aligned to yield 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.). It is also possible to use the PAM120 weight residue table using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)) integrated into the ALIGN program (version 2.0). , a gap length penalty of 12 and a gap penalty of 4 to determine the percent identity between two amino acid sequences. Alternatively, the Needleman and Wunsch (J MoI Biol. 48:444-453 (1970)) algorithm can be used using the Blossum 62 matrix or PAM250 matrix with a gap weight of 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1, 2, 3, 4, 5 or 6 to determine the percent identity between two amino acid sequences .
载体carrier
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。The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it is linked. Vectors include, but are not limited to, single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules including one or more free ends, no free ends (eg, circular); including DNA, RNA, or both. Nucleic acid molecules; and a wide variety of other polynucleotides known in the art. The vector can be introduced into the host cell through transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell. A vector can be introduced into a host cell to thereby produce a transcript, protein, or peptide, including from a protein, fusion protein, isolated nucleic acid molecule, etc. as described herein (e.g., a CRISPR transcript, such as a nucleic acid transcript , protein, or enzyme). A vector can contain a variety of expression-controlling elements, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain an origin of replication site.
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***另外的DNA片段的环状双链DNA环。One type of vector is a "plasmid," which refers to a circular double-stranded DNA circle into which additional DNA segments can be inserted, such as by standard molecular cloning techniques.
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。Another type of vector is a viral vector, in which virus-derived DNA or RNA sequences are present in packaging viruses (e.g., retroviruses, replication-deficient retroviruses, adenoviruses, replication-deficient adenoviruses, and adeno-associated virus) vector. Viral vectors also contain polynucleotides carried by the virus for transfection into a host cell. Certain vectors (eg, bacterial vectors with bacterial origins of replication and episomal mammalian vectors) are capable of autonomous replication in the host cells into which they are introduced.
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”。Other vectors (eg, non-episomal mammalian vectors) integrate into the genome of a host cell upon introduction to the host cell and are thereby replicated with the host genome. Furthermore, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "expression vectors."
宿主细胞host cell
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。 As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, which includes, but is not limited to, prokaryotic cells such as E. coli or Bacillus subtilis, such as microbial cells, fungal cells, animal cells, and plants. eukaryotic cells.
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。Those skilled in the art will understand that the design of the expression vector may depend on factors such as the choice of host cell to be transformed, the desired level of expression, and the like.
调控元件regulatory elements
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)。As used herein, the term "regulatory element" is intended to include promoters, enhancers, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and PolyU sequence), for detailed description please refer to Goeddel, "GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY" 185, Academic Press, San Diego , California (1990). In some cases, regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host cells as well as those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). Tissue-specific promoters may primarily direct expression in the desired tissue of interest, such as muscle, neurons, bone, skin, blood, a specific organ (e.g., liver, pancreas), or a specific cell type (e.g., lymphocytes). In some cases, regulatory elements may also direct expression in a timing-dependent manner (eg, in a cell cycle-dependent or developmental stage-dependent manner), which may or may not be tissue or cell type specific. In some cases, the term "regulatory element" encompasses enhancer elements such as WPRE; CMV enhancer; the R-U 5' fragment in the LTR of HTLV-I ((Mol. Cell. Biol., pp. 8( 1), pp. 466-472, 1988); the SV40 enhancer; and the intron sequence between exons 2 and 3 of rabbit β-globin (Proc. Natl. Acad. Sci. USA., Volume 78(3), pages 1527-31, 1981).
启动子Promoter
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。As used herein, the term "promoter" has a meaning known to those skilled in the art, which refers to a non-coding nucleotide sequence located upstream of a gene that can initiate the expression of a downstream gene. A constitutive promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining the gene product, results in the gene product in the cell under most or all physiological conditions of the cell. of production. An inducible promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, results in essentially only when an inducer corresponding to said promoter is present in the cell. The gene product is produced within the cell. A tissue-specific promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, results in essentially only cells of the tissue type to which the promoter corresponds. Gene products are produced in cells.
NLSNLS
“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。A "nuclear localization signal" or "nuclear localization sequence" (NLS) is an amino acid sequence that "tags" a protein for import into the nucleus via nuclear transport, ie, proteins with an NLS are transported to the nucleus. Typically, NLS contain positively charged Lys or Arg residues exposed on the protein surface. Exemplary nuclear localization sequences include, but are not limited to, NLS from: SV40 large T antigen, EGL-13, c-Myc, and TUS proteins. In some embodiments, the NLS includes the PKKKRKV sequence. In some embodiments, the NLS includes the sequence AVKRPAATKKAGQAKKKKLD. In some embodiments, the NLS includes the PAAKRVKLD sequence. In some embodiments, the NLS includes the sequence MSRRRKANPTKLSENAKKLAKEVEN. In some embodiments, the NLS includes the KLKIKRPVK sequence. Other nuclear localization sequences include, but are not limited to, the acidic M9 domain of hnRNP A1, the sequences KIPIK and PY-NLS in the yeast transcriptional repressor Matα2.
可操作地连接operably connected
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***中或当该载体被引入到宿主细胞中时，处于该宿主细胞中)。As used herein, the term "operably linked" is intended to mean that a 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).
互补性complementarity
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。As used herein, the term "complementarity" refers to the ability of a nucleic acid to form one or more hydrogen bonds with another nucleic acid sequence by means of traditional Watson-Crick or other non-traditional types. Percent complementarity represents the percentage of residues (e.g., out of 10) in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence. 5, 6, 7, 8, 9, and 10 of them are 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Perfectly complementary" means that all contiguous residues of one nucleic acid sequence form hydrogen bonds with the same number of contiguous residues of a second nucleic acid sequence. As used herein, "substantially complementary" means that in a system having 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% complementarity, or refers to two nucleic acids that hybridize under stringent conditions.
严格条件strict conditions
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。As used herein, "stringent conditions" for hybridization refer to conditions under which a nucleic acid having complementarity to a target sequence hybridizes primarily to the target sequence and does not substantially hybridize to non-target sequences. Stringent conditions are often sequence dependent and vary depending on many factors. In general, the longer the sequence, the higher the temperature at which the sequence will hybridize specifically to its target sequence.
杂交hybridization
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”。The term "hybrid" or "complementary" or "substantially complementary" means that a nucleic acid (e.g., RNA, DNA) contains nucleotide sequences that enable it to bind non-covalently, i.e., in a sequence-specific, antiparallel manner ( That is, a nucleic acid specifically binds a complementary nucleic acid) to form base pairs and/or G/U base pairs, "annealing" or "hybridization" with another nucleic acid.
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)。Hybridization requires that the two nucleic acids contain complementary sequences, although mismatches between bases may exist. 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 (e.g., 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).
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％。It will be understood that the sequence of a polynucleotide does not need to be 100% complementary to the sequence of its target nucleic acid in order to hybridize specifically. The polynucleotide may comprise 60% or higher, 65% or higher, 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or Higher, 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%.
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。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 and form a complex; or there are at least 12, 15, 16, 17, 18, 19, 20 nucleic acid sequences representing the target sequence and gRNA. One, 21, 22 or more bases can complement each other and hybridize to form a complex.
表达Express
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。As used herein, the term "expression" refers to the process by which a DNA template is transcribed into a polynucleotide (eg, into mRNA or other RNA transcript) and/or the transcribed mRNA is subsequently translated into a peptide, The process of polypeptide or protein. Transcripts and encoded polypeptides may collectively be referred to as "gene products." If the polynucleotide is derived from genomic DNA, expression can include splicing of mRNA in eukaryotic cells.
接头Connector
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)。As used herein, the term "linker" refers to a linear polypeptide formed by multiple amino acid residues linked by peptide bonds. The linker of the present invention can be a synthetic amino acid sequence or a naturally occurring polypeptide sequence, such as a polypeptide with hinge region function. Such linker polypeptides are well known in the art (see, e.g., Holliger, P. et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R.J. et al. (1994) Structure 2:1121- 1123).
治疗treat
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。As used herein, the term "treating" means treating or curing a condition, delaying the onset of symptoms of a condition, and/or delaying the progression of a condition.
受试者Subject
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。 As used herein, the term "subject" includes, but is not limited to, various animals, plants, and microorganisms.
动物animal
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)。For example, mammals, such as bovines, equids, ovines, porcines, canines, felines, leporids, rodents (e.g., mice or rats), non-human primates animal (e.g., macaque or cynomolgus monkey) or human. In certain embodiments, the subject (eg, a human) has a disorder (eg, a disorder resulting from a defect in a disease-associated gene).
植物plant
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、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。The term "plant" is understood to mean any differentiated multicellular organism capable of photosynthesis and includes crop plants at any stage of maturity or development, in particular monocots or dicots, vegetable crops including artichokes, kohlrabi, Arugula, leeks, asparagus, lettuce (e.g., head, leaf, romaine), bok choy, yam, melons (e.g., melon, watermelon, crenshaw ), honeydew, romaine melon), rapeseed crops (e.g. Brussels sprouts, cabbage, cauliflower, broccoli, kale, kale, Chinese cabbage, bok choy), cardoons, carrots, napa, Okra, onions, celery, parsley, chickpeas, parsnips, chicory, peppers, potatoes, gourds (e.g., zucchini, cucumbers, courgettes, pumpkins, pumpkins), radishes, dried onions, rutabaga, Purple eggplant (also called eggplant), salsify, endive, shallots, chicory, garlic, spinach, green onions, pumpkin, greens, beets (sugar beets and fodder beets), sweet potatoes, chard, Wasabi, tomatoes, turnips, and spices; fruit and/or vine crops such as apples, apricots, cherries, nectarines, peaches, pears, plums, prunes, cherries, quinces, almonds, chestnuts, hazelnuts, pecans, Pistachios, walnuts, citrus, blueberries, boysenberries, cranberries, currants, loganberries, raspberries, strawberries, blackberries, grapes, avocados, bananas, kiwis, persimmons, pomegranates, pineapples , tropical fruits, pome fruits, melons, mangoes, papayas, and lychees; field crops, such as clover, alfalfa, evening primrose, alfalfa, corn/maize (feed corn, sweet corn, popcorn), hops, lotus root Barley, peanuts, rice, safflower, small cereal crops (barley, oats, rye, wheat, etc.), sorghum, tobacco, kapok, leguminous plants (beans, lentils, peas, soybeans), oil-bearing plants (rapeseed, rape, etc.) mustard, poppy, olive, sunflower, coconut, castor plant, cocoa beans, groundnut), Arabidopsis, fiber plants (cotton, flax, jute), Lauraceae (cinnamon, camphor), or a plant such as coffee , sugar cane, tea, and natural rubber plants; and/or bedding plants, such as flowering plants, cacti, succulents and/or ornamental plants, and trees such as forests (broadleaf and evergreen trees, such as conifers), fruit trees, ornamental trees, and nut-bearing trees, as well as shrubs and other seedlings.
发明的有益效果Beneficial effects of the invention
本发明通过突变提高了Cas12j19蛋白的活性，具有广泛的应用前景。The present invention improves the activity of Cas12j19 protein through mutation and has broad application prospects.
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。The embodiments of the present invention will be described in detail below with reference to the drawings and examples, but those skilled in the art will understand that the following drawings and examples are only used to illustrate the present invention and do not limit the scope of the present invention. Various objects and advantageous aspects of the present invention will become apparent to those skilled in the art from the accompanying drawings and the following detailed description of preferred embodiments.
附图说明Description of drawings
图1.单位点氨基酸突变Cas蛋白编辑效率的验证结果。Figure 1. Verification results of single-site amino acid mutation Cas protein editing efficiency.
图2.突变后的E100R蛋白与野生型Cas12j19在大豆中编辑效率的验证结果。横坐标代表不同的靶点位置，纵坐标代表编辑效率。Figure 2. Verification results of the editing efficiency of mutated E100R protein and wild-type Cas12j19 in soybean. The abscissa represents different target positions, and the ordinate represents editing efficiency.
图3.单位点氨基酸饱和突变的Cas蛋白编辑效率的验证结果。 Figure 3. Verification results of Cas protein editing efficiency of single-site amino acid saturation mutation.
具体实施方式Detailed ways
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))。The following examples are only used to describe the present invention but not to limit the present invention. Unless otherwise indicated, the experiments and methods described in the examples were performed essentially according to conventional methods well known in the art and described in various references. For example, for conventional techniques such as immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA used in the present invention, see Sambrook, Fritsch ) and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd ed. (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY ) (edited by F.M. Ausubel et al., (1987)); "METHODS IN ENZYMOLOGY" series (Academic Publishing Company): "PCR 2: A PRACTICAL APPROACH" ) (edited by M.J. MacPherson, B.D. Hames and G.R. Taylor (1995)), Harlow and Lane (1988) "Antibodies: "ANTIBODIES, A LABORATORY MANUAL", and "ANIMAL CELL CULTURE" (edited by R.I. Freshney (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。In addition, if the specific conditions are not specified in the examples, the conventional conditions or the conditions recommended by the manufacturer shall be followed. If the manufacturer of the reagents or instruments used is not indicated, they are all conventional products that can be purchased commercially. Those skilled in the art will appreciate that the examples describe the invention by way of example and are not intended to limit the scope of the invention as claimed. All publications and other references mentioned herein are incorporated by reference in their entirety.
实施例1.Cas突变蛋白的获得Example 1. Obtaining Cas mutant protein
针对已知的Cas蛋白(CN111770992B中的Cas12j.19，本实施例中，将其称之为Cas12i19)，申请人通过生物信息学预测可能影响其生物学功能的关键氨基酸位点，并将氨基酸位点进行突变，得到了编辑活性提高的Cas突变蛋白。具体的，将Cas12j19编码序列经过密码子优化并合成，野生型Cas12j19的氨基酸序列如SEQ ID No.1所示，其核酸序列如SEQ ID No.2所示，通过生物信息学方法对潜在的Cas12j19与目标序列相互结合的氨基酸进行定点突变。For the known Cas protein (Cas12j.19 in CN111770992B, in this example, it is called Cas12i19), the applicant used bioinformatics to predict the key amino acid positions that may affect its biological function, and assigned the amino acid positions to Point mutations were performed to obtain Cas mutant proteins with improved editing activity. Specifically, the Cas12j19 coding sequence was codon optimized and synthesized. The amino acid sequence of wild-type Cas12j19 is shown in SEQ ID No. 1, and its nucleic acid sequence is shown in SEQ ID No. 2. The potential Cas12j19 was analyzed through bioinformatics methods. Site-directed mutagenesis of amino acids that bind to the target sequence.
SEQ ID No.1：
SEQ ID No.1:
SEQ ID No.2：
SEQ ID No.2:
通过基于PCR的定点诱变产生Cas蛋白的变体。具体的方法是以突变的位点为中心将Cas12j19蛋白的DNA序列设计分成两部分，设计两对引物分别扩增这两部分DNA序列，同时引物上引入需要突变的序列，最后通过Gibson克隆的方式将两个片段装载到pcDNA3.3-eGFP载体上。突变体的组合则通过将Cas12j19蛋白的DNA拆分成多段，使用PCR、Gibson clone实现构建。片段扩增试剂盒：TransStart FastPfu DNA Polymerase(含2.5mM dNTPs)，具体实验流程详见说明书。胶回收试剂盒：Gel DNA Extraction Mini Kit，具体实验流程详见说明书。载体构建所用试剂盒：pEASY-Basic Seamless Cloning and Assembly Kit(CU201-03)，具体实验流程详见说明书。所涉及的突变氨基酸位点以及所采用的引物序列如下表所示：

Variants of Cas proteins were generated by PCR-based site-directed mutagenesis. The specific method is to divide the DNA sequence design of Cas12j19 protein into two parts centered on the mutation site, design two pairs of primers to amplify the two parts of the DNA sequence respectively, and introduce the sequence that needs to be mutated on the primers, and finally use Gibson cloning. Load both fragments into pcDNA3.3-eGFP vector. The combination of mutants is constructed by splitting the DNA of the Cas12j19 protein into multiple segments and using PCR and Gibson clone. Fragment amplification kit: TransStart FastPfu DNA Polymerase (containing 2.5mM dNTPs), please refer to the instruction manual for specific experimental procedures. Glue recovery kit: Gel DNA Extraction Mini Kit, please refer to the instruction manual for detailed experimental procedures. The kit used for vector construction: pEASY-Basic Seamless Cloning and Assembly Kit (CU201-03). Please refer to the instruction manual for specific experimental procedures. The mutated amino acid sites involved and the primer sequences used are shown in the following table:

基于上述氨基酸突变位点，分别获得了Cas12j19的野生型蛋白(WT)，以及上述氨基酸单个位点发生突变的蛋白(以突变类型命名)：D50R、E100R和N103D，其相对于SEQ ID No.1所示的序列，自N端起第50位氨基酸突变为R，自N端起第100位氨基酸突变为R，自N端起第103位氨基酸突变为D。Based on the above amino acid mutation sites, the wild-type protein (WT) of Cas12j19 and the proteins with mutations in the above amino acid single sites (named after the mutation type) were obtained: D50R, E100R and N103D, which are relative to SEQ ID No.1 In the sequence shown, the 50th amino acid from the N terminus is mutated to R, the 100th amino acid from the N terminus is mutated to R, and the 103rd amino acid from the N terminus is mutated to D.
实施例2.Cas突变蛋白的编辑活性的验证Example 2. Verification of editing activity of Cas mutant protein
采用实施例1获得的不同的Cas蛋白在动物细胞中验证其基因编辑的活性，针对中国仓鼠卵巢细胞(CHO)FUT8基因设计靶点，FUT8-3：ATGGAGGCTGTCTACAATGGGG A，斜体部分为PAM序列，其余区域为靶向区。载体pcDNA3.3经改造后带有EGFP荧光蛋白及PuroR抗性基因。经酶切位点XbaI和PstI***SV40NLS-Cas-XX融合蛋白；经酶切位点Mfe1***U6启动子及gRNA序列。CMV启动子启动融合蛋白SV40NLS-Ca s-XX-NLS-GFP表达。蛋白Cas-XX-NLS与蛋白GFP用连接肽T2A进行连接。启动子EF-1α启动嘌呤霉素抗性基因表达。铺板：CHO细胞融合度至70-80％进行铺板，12孔板中接种细胞数为8*10^4细胞/孔。转染：铺板24h进行转染，100μl opti-MEM中加入6.25μl Hieff TransTM脂质体核酸转染试剂，混匀；100μl opti-MEM中加入2.5ug质粒，混匀。稀释好的Hieff TransTM脂质体核酸转染试剂与稀释后的质粒混合均匀，室温孵育20min。孵育好的混合液加入铺有细胞的培养基中进行转染。转染48h后，用胰蛋白酶-EDTA(0.05％)消化，用流式细胞仪(FACS)分选具有GFP信号的细胞。Different Cas proteins obtained in Example 1 were used to verify their gene editing activity in animal cells, and the target was designed for the Chinese hamster ovary cell (CHO) FUT8 gene, FUT8-3: ATGGAGGCTGTCTACAATGGGG A, the italicized part is the PAM sequence, and the remaining regions is the target area. The vector pcDNA3.3 was modified to contain EGFP fluorescent protein and PuroR resistance gene. The SV40NLS-Cas-XX fusion protein was inserted through the restriction site XbaI and PstI; the U6 promoter and gRNA sequence were inserted through the restriction site Mfe1. The CMV promoter drives the expression of the fusion protein SV40NLS-Ca s-XX-NLS-GFP. The protein Cas-XX-NLS and the protein GFP are connected using the connecting peptide T2A. Promoter EF-1α drives puromycin resistance gene expression. Plating: Plate the CHO cells until the confluence is 70-80%. The number of cells seeded in the 12-well plate is 8*10^4 cells/well. Transfection: Plate for 24 hours for transfection. Add 6.25 μl Hieff Trans TM liposome nucleic acid transfection reagent to 100 μl opti-MEM and mix well. Add 2.5ug plasmid to 100 μl opti-MEM and mix well. Mix the diluted Hieff Trans TM liposome nucleic acid transfection reagent and the diluted plasmid evenly, and incubate at room temperature for 20 minutes. The incubated mixture was added to the culture medium on which cells were plated for transfection. After 48 hours of transfection, the cells were digested with trypsin-EDTA (0.05%), and cells with GFP signal were sorted using flow cytometry (FACS).
提DNA、PCR扩增编辑区附近、送hiTOM测序：细胞经胰酶消化处理后进行收集，经细胞/组织基因组DNA提取试剂盒(百泰克)进行基因组DNA提取。对基因组DNA扩增靶点附近区域。PCR产物进行hiTOM测序。测序数据分析，统计靶点位置上游15n t、下游10nt范围内的序列种类及比例，统计序列中SNV频率大于/等于1％或非SNV的突变频率大于/等于0.06％的序列，得到Cas-XX蛋白对靶点位置的编辑效率。CHO细胞F UT8基因靶点序列：FUT8-Cas-XX-g3：ATGGAGGCTGTCTACAATGGGGA，斜体部分为PAM序列，其余区域为靶向区。gRNA序列为： 加粗区域为靶向区，其他区域为DR(同向重复序列)区。Extract DNA, PCR amplify near the editing area, and send to hiTOM for sequencing: cells are collected after trypsin digestion, and genomic DNA is extracted using a cell/tissue genomic DNA extraction kit (Biotech). Amplify the region near the target site from genomic DNA. PCR products were subjected to hiTOM sequencing. Sequencing data analysis, counting the sequence types and proportions within the range of 15nt upstream and 10nt downstream of the target position, counting the sequences with SNV frequency greater than/equal to 1% or non-SNV mutation frequency greater than/equal to 0.06%, to obtain Cas-XX The editing efficiency of the protein at the target position. CHO cell F UT8 gene target sequence: FUT8-Cas-XX-g3: ATGGAGGCTGTCTACAATGGGGA, the italicized part is the PAM sequence, and the rest of the region is the targeting region. The gRNA sequence is: The bolded region is the targeting region, and the other regions are DR (direct repeat sequence) regions.
图1示出了野生型Cas12j19蛋白(WT，SEQ ID No.1所示)以及单个氨基酸位点突变的突变蛋白的编辑活性。如图1，与WT相比，E100R氨基酸位点的突变能够显著提高Cas12j19蛋白的编辑效率，而D50R和N103D的突变则会降低Cas12j19蛋白的编辑效率。Figure 1 shows the editing activity of wild-type Cas12j19 protein (WT, shown in SEQ ID No. 1) and mutant proteins with single amino acid site mutations. As shown in Figure 1, compared with WT, mutations at the E100R amino acid site can significantly improve the editing efficiency of Cas12j19 protein, while mutations in D50R and N103D will reduce the editing efficiency of Cas12j19 protein.
实施例3.Cas突变蛋白在大豆中编辑活性的验证Example 3. Verification of editing activity of Cas mutant protein in soybean
对大豆基因组中FAD基因以及BADH基因分别设计多个靶点，靶点信息如表2所示，将设计的靶点分别构入含有实施例1中的Cas突变蛋白E100R的载体中，为验证载体的编辑效率，将构建完成的载体进行大豆幼苗的发根农杆菌瞬时转化。Multiple targets were designed for the FAD gene and BADH gene in the soybean genome. The target information is shown in Table 2. The designed targets were constructed into vectors containing the Cas mutant protein E100R in Example 1, which were used as verification vectors. With high editing efficiency, the constructed vector was used for transient transformation of soybean seedlings with Agrobacterium rhizogenes.
(1)大豆种子萌发：取营养土及蛭石按2:1的比例混匀，平铺在穴盘上，将大豆种 子以0.5cm的深度点播到穴盘中，浇适量水准备萌发。(1) Soybean seed germination: Mix nutrient soil and vermiculite in a ratio of 2:1, spread it flat on the plug tray, and place the soybean seeds. Sow the seeds into the plug tray at a depth of 0.5cm, pour an appropriate amount of water to prepare for germination.
(2)菌板及菌液的准备：取500～600μl发根农杆菌K599甘油菌液涂板，同时取50μl菌液加入含有相应抗生素的TY培养液，28℃过夜培养。(2) Preparation of bacterial plate and bacterial liquid: Take 500-600 μl of Agrobacterium rhizogenes K599 glycerol bacterial liquid and apply it on the plate. At the same time, take 50 μl of bacterial liquid and add TY culture medium containing the corresponding antibiotics, and culture at 28°C overnight.
(3)发根农杆菌侵染大豆：用刀片在生长7-14天的大豆幼苗子叶的下胚轴1cm处切一倾斜的伤口，伤口处蘸取接种发根农杆菌。然后将接种的幼苗的下胚轴直接种植在潮湿的无菌蛭石培养盒中，每棵浇10ml K599侵染悬浮液，随后在16天内保持高湿度，以促进大豆毛状根生长。(3) Agrobacterium rhizogenes infects soybeans: Use a blade to cut an inclined wound 1cm from the hypocotyls of the cotyledons of soybean seedlings that have grown for 7-14 days, and dip the wound into the wound to inoculate it with Agrobacterium rhizogenes. The hypocotyls of the inoculated seedlings were then directly planted in a humid sterile vermiculite culture box, and each plant was poured with 10 ml of K599 infection suspension, and then maintained at high humidity for 16 days to promote soybean hairy root growth.
毛状根长出后，利用TPS法提取大豆毛状根的DNA，分别用检测引物扩增相应片段，引物信息见表3，扩增产物经sanger法进行测序，根据测序结果确定编辑效率。After the hairy roots grow, the DNA of the soybean hairy roots is extracted using the TPS method, and the corresponding fragments are amplified using detection primers. The primer information is shown in Table 3. The amplified products are sequenced by the Sanger method, and the editing efficiency is determined based on the sequencing results.
表2.靶点的序列信息
Table 2. Sequence information of targets
表3.检测引物的序列信息
Table 3. Sequence information of detection primers
结果如图2所示，检测到野生型Cas12j19与突变后的E100R在不同靶点处的编辑效率明显不同，突变后的E100R蛋白在靶点BADH2-6，FAD-1A-1，BADH1-5，FAD-1B-2，FAD-1B-3表现出显著的编辑活性，野生型Cas12j19在各靶点处均未检测到编辑活性；突变后的E100R蛋白与野生型Cas12j19相比在各靶点的编辑效率均显著提高。The results are shown in Figure 2. It was detected that the editing efficiencies of wild-type Cas12j19 and mutated E100R were significantly different at different target sites. The mutated E100R protein was detected at target sites BADH2-6, FAD-1A-1, BADH1-5, FAD-1B-2 and FAD-1B-3 showed significant editing activity, and wild-type Cas12j19 had no editing activity detected at each target site; compared with wild-type Cas12j19, the mutated E100R protein edited at each target site Efficiencies are significantly improved.
实施例4.对野生型Cas12j19第100位进行饱和突变并验证其编辑活性Example 4. Perform saturation mutation on position 100 of wild-type Cas12j19 and verify its editing activity
以野生型Cas12j19蛋白为模板，参考实施例1中获得突变蛋白的方法，设计相应的点突引物，通过基于PCR的定点诱变产生Cas蛋白的变体，获得第100位突变为多种氨基酸类型(A，V，G，L，D，F，W，Y，N，S，Q，K，M，T，C，P，H，或I)的Cas蛋白变体。Using the wild-type Cas12j19 protein as a template, refer to the method for obtaining the mutant protein in Example 1, design corresponding point primers, generate variants of the Cas protein through site-directed mutagenesis based on PCR, and obtain mutations at position 100 into multiple amino acid types. Cas protein variants of (A, V, G, L, D, F, W, Y, N, S, Q, K, M, T, C, P, H, or I).
采用实施例2中验证不同Cas蛋白在动物细胞中编辑活性的方法，验证上述各Cas蛋白变体在动物细胞的活性。结果如图3所示，对野生型Cas蛋白第100位氨基酸位点进行饱和突变，突变后的氨基酸类型不同对于动物细胞的编辑活性也不相同，其中第100位突变为K，Y，M，F，W，S，H，V，I，N时，Cas蛋白变体的编辑效率显著高于野生型Cas12j19，氨基酸突变为C或L后与野生型Cas12j19活性基本一致，氨基酸突变为Q， G，T，D，P后编辑活性低于野生型Cas12j19，氨基酸类型突变为A时，Cas蛋白变体失去编辑活性。The method of verifying the editing activities of different Cas proteins in animal cells in Example 2 was used to verify the activity of each of the above Cas protein variants in animal cells. The results are shown in Figure 3. A saturation mutation was performed on the 100th amino acid position of the wild-type Cas protein. Different types of amino acids after mutation have different editing activities in animal cells. The 100th position was mutated to K, Y, M. When F, W, S, H, V, I, N, the editing efficiency of the Cas protein variant is significantly higher than that of wild-type Cas12j19. After the amino acid mutation is C or L, the activity is basically the same as that of wild-type Cas12j19. When the amino acid mutation is Q, The editing activity after G, T, D, and P is lower than that of wild-type Cas12j19. When the amino acid type is mutated to A, the Cas protein variant loses its editing activity.
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。 Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all teachings that have been published, and these changes are within the protection scope of the present invention. . The full scope of the present invention is given by the appended claims and any equivalents thereof.

Claims (15)

  1. 一种Cas突变蛋白，所述突变蛋白与亲本Cas蛋白的氨基酸序列相比，在对应于SEQ ID No.1所示氨基酸序列的以下氨基酸位点处存在突变：第100位；优选的，所述第100位氨基酸突变为K，Y，M，F，R，W，S，H，V，I，N，C，L中的任意一种。A Cas mutant protein, compared with the amino acid sequence of the parent Cas protein, there is a mutation at the following amino acid position corresponding to the amino acid sequence shown in SEQ ID No. 1: No. 100; preferably, the The 100th amino acid is mutated to any one of K, Y, M, F, R, W, S, H, V, I, N, C, and L.
  2. 根据权利要求1所述的Cas突变蛋白，其特征在于，所述亲本Cas蛋白为Cas12j家族蛋白。The Cas mutant protein according to claim 1, wherein the parent Cas protein is a Cas12j family protein.
  3. 根据权利要求1或2所述的Cas突变蛋白，其特征在于，所述Cas突变蛋白选自以下I-III任意一组：The Cas mutant protein according to claim 1 or 2, characterized in that the Cas mutant protein is selected from any group of the following I-III:
    I、由SEQ ID No.1所示氨基酸序列在包含以下氨基酸位点处产生突变得到的Cas突变蛋白：第100位氨基酸；I. Cas mutant protein obtained by mutating the amino acid sequence shown in SEQ ID No. 1 at the following amino acid positions: the 100th amino acid;
    II、与I所述的Cas突变蛋白相比，具有I中所述的突变位点；并且，与I所述的Cas突变蛋白相比，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％、至少99％、至少99.1％、至少99.2％、至少99.3％、至少99.4％、至少99.5％、至少99.6％、至少99.7％、至少99.8％、或至少99.9％的序列同一性的Cas突变蛋白；II. Compared with the Cas mutant protein described in I, it has the mutation site described in I; and, compared with the Cas mutant protein described in I, it has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4% , a Cas mutant protein with at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity;
    III、与I所述的Cas突变蛋白相比，具有I中所述的突变位点；并且，与I所述的Cas突变蛋白相比，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。III. Compared with the Cas mutant protein described in I, it has the mutation site described in I; and, compared with the Cas mutant protein described in I, it has a sequence with one or more amino acid substitutions, deletions or additions. ; The one or more amino acids include substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids.
  4. 一种融合蛋白，所述融合蛋白包括权利要求1-3任一所述的Cas突变蛋白以及其他的修饰部分；优选的，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。A fusion protein, the fusion protein includes the Cas mutant protein according to any one of claims 1-3 and other modified parts; preferably, the modified part is selected from another protein or polypeptide, a detectable label or its random combination.
  5. 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1-3任一所述Cas突变蛋白的多核苷酸序列，或编码权利要求4所述融合蛋白的多核苷酸序列。An isolated polynucleotide, characterized in that the polynucleotide is a polynucleotide sequence encoding the Cas mutant protein of any one of claims 1-3, or a polynucleotide encoding the fusion protein of claim 4 sequence.
  6. 一种载体，其特征在于，所述载体包含权利要求5所述的多核苷酸以及与之可操作连接的调控元件。A vector, characterized in that the vector contains the polynucleotide of claim 5 and a regulatory element operably connected thereto.
  7. 一种CRISPR-Cas***，其特征在于，所述***包括权利要求1-3任一所述的Cas突变蛋白以及至少一种gRNA；A CRISPR-Cas system, characterized in that the system includes the Cas mutant protein according to any one of claims 1-3 and at least one gRNA;
    所述gRNA能够结合权利要求1-3任一所述的Cas突变蛋白。The gRNA is capable of binding to the Cas mutant protein of any one of claims 1-3.
  8. 一种组合物，其特征在于，所述组合物包含：A composition, characterized in that the composition contains:
    (i)蛋白组分，其选自：权利要求1-3任一所述的Cas突变蛋白或权利要求4所述的融合蛋白；(i) Protein component, which is selected from: the Cas mutant protein described in any one of claims 1-3 or the fusion protein described in claim 4;
    (ii)核酸组分，其为gRNA，所述gRNA能够结合权利要求1-3任一所述的Cas突变蛋白；(ii) a nucleic acid component, which is gRNA, and the gRNA is capable of binding to the Cas mutant protein of any one of claims 1-3;
    所述蛋白组分与核酸组分相互结合形成复合物。The protein component and the nucleic acid component combine with each other to form a complex.
  9. 一种活化的CRISPR复合物，所述活化的CRISPR复合物包含：An activated CRISPR complex comprising:
    (i)蛋白组分，其选自：权利要求1-3任一所述的Cas突变蛋白或权利要求4所述的融合蛋白；(i) Protein component, which is selected from: the Cas mutant protein described in any one of claims 1-3 or the fusion protein described in claim 4;
    (ii)核酸组分，其为gRNA，所述gRNA包括能够结合权利要求1-3任一所述的Cas突变蛋白的同向重复序列和能够靶向靶序列的引导序列；(ii) a nucleic acid component, which is a gRNA, which includes a direct repeat sequence capable of binding the Cas mutant protein of any one of claims 1-3 and a guide sequence capable of targeting a target sequence;
    (iii)结合在(ii)中所述gRNA上的靶序列。(iii) Binding to a target sequence on the gRNA described in (ii).
  10. 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1-3任一所述的Cas突变蛋白，或权利要求4所述的融合蛋白，或权利要求5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***，或权利要求8所述的组合物，或权利要求9所述的活化的CRISPR复合物。An engineered host cell, characterized in that the host cell contains the Cas mutant protein of any one of claims 1-3, or the fusion protein of claim 4, or the polynucleoside of claim 5 acid, or right The vector of claim 6, or the CRISPR-Cas system of claim 7, or the composition of claim 8, or the activated CRISPR complex of claim 9.
  11. 权利要求1-3任一所述的Cas突变蛋白，或权利要求4所述的融合蛋白，或权利要求5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***，或权利要求8所述的组合物，或权利要求9所述的活化的CRISPR复合物，或权利要求10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。The Cas mutant protein of any one of claims 1-3, or the fusion protein of claim 4, or the polynucleotide of claim 5, or the vector of claim 6, or the vector of claim 7 The CRISPR-Cas system, or the composition of claim 8, or the activated CRISPR complex of claim 9, or the host cell of claim 10 in gene editing, gene targeting or gene cutting. Application; or use in the preparation of reagents or kits for gene editing, gene targeting or gene cleavage.
  12. 权利要求1-3任一所述的Cas突变蛋白，或权利要求4所述的融合蛋白，或权利要求5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***，或权利要求8所述的组合物，或权利要求9所述的活化的CRISPR复合物，或权利要求10所述的宿主细胞在选自如下任一或任意几种中的应用：The Cas mutant protein of any one of claims 1-3, or the fusion protein of claim 4, or the polynucleotide of claim 5, or the vector of claim 6, or the vector of claim 7 The CRISPR-Cas system, or the composition of claim 8, or the activated CRISPR complex of claim 9, or the host cell of claim 10 is selected from any one or any several of the following: application:
    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。Targeting and/or editing of target nucleic acids; cleavage of double-stranded DNA, single-stranded DNA or single-stranded RNA; non-specific cleavage and/or degradation of side nucleic acids; non-specific cleavage of single-stranded nucleic acids; nucleic acid detection; specific editing of double-stranded nucleic acids ;Base editing double-stranded nucleic acid;Base editing single-stranded nucleic acid.
  13. 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1-3任一所述的Cas突变蛋白，或权利要求4所述的融合蛋白，或权利要求5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***，或权利要求8所述的组合物，或权利要求9所述的活化的CRISPR复合物，或权利要求10所述的宿主细胞进行接触。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 Cas mutant protein of any one of claims 1-3, or the fusion protein of claim 4, or The polynucleotide of claim 5, or the vector of claim 6, or the CRISPR-Cas system of claim 7, or the composition of claim 8, or the activated CRISPR-Cas system of claim 9 The CRISPR complex, or the host cell of claim 10 is contacted.
  14. 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1-3任一所述的Cas突变蛋白，或权利要求4所述的融合蛋白，或权利要求5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***，或权利要求8所述的组合物，或权利要求9所述的活化的CRISPR复合物，或权利要求10所述的宿主细胞。A kit for gene editing, gene targeting or gene cutting, the kit comprising the Cas mutant protein of any one of claims 1-3, or the fusion protein of claim 4, or claim 5 The polynucleotide, or the vector of claim 6, or the CRISPR-Cas system of claim 7, or the composition of claim 8, or the activated CRISPR complex of claim 9 , or the host cell of claim 10.
  15. 权利要求1-3任一所述的Cas突变蛋白，或权利要求4所述的融合蛋白，或权利要求5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***，或权利要求8所述的组合物，或权利要求9所述的活化的CRISPR复合物，或权利要求10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：The Cas mutant protein of any one of claims 1-3, or the fusion protein of claim 4, or the polynucleotide of claim 5, or the vector of claim 6, or the vector of claim 7 The CRISPR-Cas system, or the composition of claim 8, or the activated CRISPR complex of claim 9, or the use of the host cell of claim 10 in preparing a preparation or kit, Preparations or kits for:
    (i)基因或基因组编辑；(i) Gene or genome editing;
    (ii)靶核酸检测和/或诊断；(ii) Target nucleic acid detection and/or diagnosis;
    (iii)编辑靶基因座中的靶序列来修饰生物；(iii) Edit target sequences in target loci to modify organisms;
    (iv)疾病的治疗；(iv) Treatment of disease;
    (v)靶向靶基因；(v) targeting target genes;
    (vi)切割目的基因。 (vi) Cut the target gene.
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