WO2024046307A1 - 一种突变的v型crispr酶及其应用 - Google Patents

一种突变的v型crispr酶及其应用 Download PDF

Info

Publication number
WO2024046307A1
WO2024046307A1 PCT/CN2023/115471 CN2023115471W WO2024046307A1 WO 2024046307 A1 WO2024046307 A1 WO 2024046307A1 CN 2023115471 W CN2023115471 W CN 2023115471W WO 2024046307 A1 WO2024046307 A1 WO 2024046307A1
Authority
WO
WIPO (PCT)
Prior art keywords
amino acid
seq
protein
cas12 protein
cas12
Prior art date
Application number
PCT/CN2023/115471
Other languages
English (en)
French (fr)
Inventor
潘伟业
孙阳
陈重建
朱鹏宇
尤胜浩
Original Assignee
北京迅识科技有限公司
浙江迅识生物科技有限公司
北京迅识生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 北京迅识科技有限公司, 浙江迅识生物科技有限公司, 北京迅识生物科技有限公司 filed Critical 北京迅识科技有限公司
Publication of WO2024046307A1 publication Critical patent/WO2024046307A1/zh

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays

Definitions

  • the present application belongs to the field of biotechnology, and specifically relates to a mutant V-type CRISPR enzyme and its application.
  • Type V CRISPR-Cas system is also called the Cas12 family. It differs from other CRISPR-Cas systems in that it is an RNA-mediated single-effector ribozyme driven by a single RuvC active center. As more and more Cas12s are discovered and identified, this family has multiple categories such as V-A, V-B, V-C, V-D, V-E, V-F, V-G, V-H, V-I, V-J, V-K, etc.
  • Cas12a and Cas12b are used in many nucleic acid detection technologies and clinical detection products combined with amplification technology due to their excellent reaction performance.
  • Cas12a or Cas12b both possess RNA-mediated cis-DNase activity and trans-DNase activity, and have emerging detection applications; although in some reports it is mentioned that Cas12a or Cas12b has RNA
  • the mediated trans-RNase activity can cleave RNA substrates with 1/10 efficiency compared to DNA substrates, but its selectivity for DNA substrates limits its further application in nucleic acid amplification systems. . This limited discrimination of one order of magnitude opens up the possibility of detection of RNA probes as another reporter substrate.
  • this application provides a mutated V-type CRISPR enzyme and its application.
  • this application involves the following aspects:
  • amino acid sequence of the reference Cas12 protein is shown in SEQ ID NO: 1.
  • the fingers upstream and downstream of the ⁇ -helix of the gatekeeping amino acids in the reference Cas12 protein were replaced with zinc finger domains.
  • the amino acid sequence of the reference Cas12 protein is shown in SEQ ID NO:1.
  • An engineered Cas12 protein or a functional derivative thereof the sequence of which is the amino acid sequence shown in any one of SEQ ID NO:5, 7, 9 or 12; or is the same as SEQ ID NO:5, 7
  • the amino acid sequence represented by any one of , 9 or 12 is an amino acid sequence having 90% or more identity.
  • a detection system for detecting target nucleic acid molecules comprising:
  • RNA which guides the Cas12 protein or its functional derivative to specifically bind to a target nucleic acid molecule; and a nucleic acid probe.
  • a method for detecting target nucleic acid molecules in a sample comprising:
  • the target nucleic acid molecule is detected by measuring a detectable signal generated by the engineered Cas12 protein or functional derivative thereof cleaving the nucleic acid probe.
  • nucleic acid probe is an RNA probe.
  • a detection system for selectively detecting target nucleic acid molecules comprising:
  • RNA which guides the Cas12 protein or its functional derivative to specifically bind to a target nucleic acid molecule
  • nucleic acid probe is a DNA probe or an RNA probe.
  • This application uses protein mutation rational mutation technology to remove the restriction of RuvC in Cas12b to recognize and cleave DNA substrates. It can specifically recognize DNA substrates or specifically recognize RNA substrates, and solve the problem of DNA/RNA substrate recognition in practical applications. The problem of insufficient differentiation.
  • the engineered Cas12 protein or its functional derivative of the present application improves the trans-cleavage activity of Cas12 and opens The latter reacts more efficiently with RNA substrates and improves the reporting efficiency of Cas12 using RNA probes.
  • Figure 1 shows the sequence alignment results of Cas12b and Cas12g
  • FIG. 1 shows the electrophoresis patterns of four mutant proteins
  • Figure 3 shows the DNA probe CRISPR cleavage results of wild-type and mutant proteins
  • Figure 4 shows the DNA probe CRISPR cleavage results of the mutant protein
  • Figure 5 shows the RNA probe CRISPR cleavage results of the mutant protein
  • Figure 6 shows the CRISPR cleavage results of the mutant protein applied to the RPA amplification reaction system.
  • Cas12 or “Cas12 protein” includes Cas12a (also known as Cpf1), Cas12b, Cas12c, Cas12d, Cas12e, Cas12h, Cas12i, Cas12g, and the like.
  • the Cas12 protein is a Cas12b protein, which is used in its broadest sense and includes a parent or reference Cas12b protein (e.g., AaCas12b with the amino acid sequence of SEQ ID NO: 1), derivatives or variants thereof bodies as well as functional fragments, such as oligonucleotide-binding fragments thereof.
  • “functional derivatives” of a protein include various variants or functional domains of the protein as long as the variants or functional domains retain the function of a functional domain of the protein ( Whether it is an enhanced function or a weakened function), it can be called a functional derivative of the protein.
  • a functional derivative of the protein For example, for the Cas12 protein, Cas12 protein variants or truncations that retain some of its domain functions are functional derivatives of the Cas12 protein.
  • domain or “protein domain” refers to a portion of a protein sequence that can exist and function independently of the remainder of the protein chain.
  • guide RNA As used herein, “guide RNA”, “sgRNA” and “gRNA” are used interchangeably herein and refer to RNA capable of forming a complex with Cas12 protein and target nucleic acid.
  • nucleic acid As used herein, the terms “nucleic acid,” “polynucleotide,” and “nucleotide sequence” are used interchangeably and refer to a polymeric form of nucleotides of any length, including deoxyribonucleotides, ribonucleotides, Combinations thereof and the like. "Oligonucleotide” and “oligonucleotide” are used interchangeably and refer to short polynucleotides of no more than about 50 nucleotides.
  • complementarity of a nucleic acid refers to the ability of one nucleic acid to form hydrogen bonds with another nucleic acid through traditional Watson-Crick base pairing. Percent complementarity represents the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (i.e., Watson-Crick base pairing) with another nucleic acid molecule (e.g., about 5, 6, 7, 8, 9, 10 out of 10 are approximately 50%, 60%, 70%, 80%, 90% and 100% complementary respectively). "Perfectly complementary” means that all contiguous residues of a nucleic acid sequence form hydrogen bonds with the same number of contiguous residues in a second nucleic acid sequence.
  • substantially complementary means at least about 70%, 75%, 80% over a region of about 40, 50, 60, 70, 80, 100, 150, 200, 250 or more nucleotides.
  • a single base or a single nucleotide according to the Watson-Crick base pairing principle, when A matches T or U, C matches G or I, it is called complementary or matching, and vice versa; and other bases Base pairs are called non-complementary or mismatched.
  • complementary in this application includes “completely complementary” and “substantially complementary”. As long as two nucleic acid sequences can form a stable hybrid double strand through Walson-Crick base pairing, the two nucleic acid sequences are said to be “complementary”, and the process of forming a stable hybrid double strand is called “complementary hybridization”.
  • wild type has the meaning commonly understood by those skilled in the art to mean the typical form of an organism, strain, or organism that distinguishes it from mutants or variants as it occurs in nature. Gene or trait. It can be isolated from natural resources and not deliberately modified.
  • nucleic acid molecule or polypeptide As used herein, the terms “non-naturally occurring” or “engineered” are used interchangeably and refer to artificial involvement. 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 of at least one other component with which it is naturally associated or naturally occurring.
  • the term "identity" is used to mean a sequence match between two polypeptides or between two nucleic acids. When a position in two compared sequences is occupied by the same base or amino acid monomer subunit (For example, if one position in each of two DNA molecules is occupied by adenine, or one position in each of two polypeptides is occupied by lysine), then each molecule is identical at that position.
  • the "percent identity” between these two sequences is a function of the number of matching positions common to both sequences divided by the number of positions being compared x 100. For example, two sequences are 60% identical if 6 out of 10 positions match. For example, the DNA sequences CTGACT and CAGGTT are 50% identical (3 matches out of 6 total positions).
  • this comparison is made when two sequences are aligned to yield maximum identity.
  • This alignment can be achieved, for example, by the method in 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.) to proceed. It is also possible to adopt the PAM 120 weighted residue table and integrate it into the ALIGN program (version 2.0) using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)). A gap length penalty of 12 and a gap penalty of 4 are used to determine the percent identity between two amino acid sequences. Additionally, the algorithm of Needleman and Wunsch (J MoI Biol.
  • variants or mutants is construed as a polynucleotide or polypeptide, respectively, that differs from a reference polynucleotide or polypeptide but retains essential properties.
  • Typical variants of a polynucleotide differ from the nucleic acid sequence of another reference polynucleotide. Changes in the variant nucleic acid sequence may or may not alter the amino acid sequence of the polypeptide encoded by the reference polynucleotide. Nucleotide changes can result in amino acid substitutions, additions, deletions, fusions, and truncations in the polypeptide encoded by the reference sequence, as described below.
  • Typical variants of a polypeptide differ in amino acid sequence from another reference polypeptide. Typically, the differences are limited such that the sequences of the reference polypeptide and the variant are very similar overall and identical in many regions.
  • the amino acid sequences of the variant and reference polypeptides may differ by any combination of one or more substitutions, additions, deletions.
  • the replaced or inserted amino acid residue may or may not be the amino acid residue encoded by the genetic code.
  • Variants of a polynucleotide or polypeptide may be naturally occurring (such as allelic variants), or may be unknown naturally occurring variants.
  • Non-naturally occurring variants of polynucleotides and polypeptides can be prepared by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to those skilled in the art.
  • target nucleic acid or “target nucleic acid molecule” refers to the target nucleic acid in the sample, which may be target RNA, target DNA, or may contain both target RNA and target. DNA.
  • This application provides an engineered Cas12 protein or functional derivative thereof, which contains the following mutations:
  • the specificity of the RuvC and/or Nuc active center in the reference Cas12 protein to recognize and cleave DNA substrates was modified.
  • the reference Cas12 protein may be a known wild-type Cas12 protein.
  • the reference Cas12 protein can be a known wild-type Cas12 protein.
  • the reference Cas12 protein can be wild-type Cas12b protein.
  • the reference Cas12 protein can be wild-type AaCas12b protein. Its amino acid sequence is shown in SEQ ID NO:1.
  • the specificity of the RuvC active center in the reference Cas12 protein to recognize and cleave DNA substrates is engineered.
  • the specificity of the Nuc active center in the reference Cas12 protein to recognize and cleave DNA substrates is modified.
  • the specificity of the RuvC and Nuc active centers in the reference Cas12 protein to recognize and cleave DNA substrates is modified.
  • modifying the specificity of the RuvC and/or Nuc active center in the reference Cas12 protein to recognize and cleave DNA substrates includes the following methods:
  • the fingers upstream and downstream of the ⁇ -helix of the gatekeeping amino acids in the reference Cas12 protein were replaced with zinc finger domains.
  • the restriction removal of the RuvC and/or Nuc active center recognition cleavage DNA substrate in the reference Cas12 protein is a domain replacement targeting part or all of the region in positions 915-945, where the amino acid position numbering is as follows Defined by SEQ ID NO:1.
  • Part of the region can be one site or multiple sites in positions 915-945, or it can be one region and multiple regions.
  • the partial region can be site P916, or site L941, or site P916 and site L941; the partial region can be positions 919-944, or positions 916-944.
  • the restriction removal of the RuvC and/or Nuc active center recognition cleavage DNA substrate in the reference Cas12 protein is a domain replacement targeting part or all of the region in positions 919-944, where the amino acid position numbering is as follows Defined by SEQ ID NO:1.
  • the restriction of the RuvC and/or Nuc active center recognition cleavage DNA substrate in the reference Cas12 protein is to replace part or all of the region in positions 916-944, for example, with a zinc finger.
  • the zinc finger domain has 1 to 2 zinc binding sites selected from the group consisting of [CxxxxC], [CxxxxH], [CxxxC], [HxxxH], [CxxC], One of [CxxH], where x represents any natural amino acid.
  • the zinc finger domain is that of Cas12g.
  • the zinc finger domain is that of HIV-1 nucleocapsid protein 7 (Ncp7).
  • positions 919-944 of the reference Cas12 protein are replaced with the zinc finger domain of Cas12g.
  • positions 919-944 of the reference Cas12 protein are replaced with the amino acid sequence shown in SEQ ID NO:4, where the amino acid position numbering is as defined in SEQ ID NO:1.
  • the amino acid sequence of the obtained engineered Cas12 protein is shown in SEQ ID NO:5.
  • positions 919-944 of the reference Cas12 protein are replaced with the zinc finger domain of Ncp7.
  • positions 919-944 of the reference Cas12 protein are replaced with the amino acid sequence shown in SEQ ID NO:6, where the amino acid position numbering is as defined in SEQ ID NO:1.
  • the amino acid sequence of the obtained engineered Cas12 protein is shown in SEQ ID NO:7.
  • the specificity of the RuvC and/or Nuc active center in the reference Cas12 protein to recognize and cleave DNA substrates is modified by replacing positions 919-944 of the gatekeeper amino acids in the reference Cas12 protein with SEQ ID NO :
  • the amino acid sequence of the obtained engineered Cas12 protein is shown in SEQ ID NO:9.
  • the specificity of the RuvC and/or Nuc active center in the reference Cas12 protein to recognize and cleave DNA substrates is modified by replacing positions 916-944 of the gatekeeper amino acids in the reference Cas12 protein with SEQ ID NO :11, the amino acid sequence shown in which the amino acid position number is as defined in SEQ ID NO:1.
  • the amino acid sequence of the obtained engineered Cas12 protein is shown in SEQ ID NO:12.
  • the present application also provides an engineered Cas12 protein or a functional derivative thereof, which contains one of the following mutations based on the reference Cas12 protein:
  • the amino acid sequence of the reference Cas12 protein is shown in SEQ ID NO:1.
  • any of the above-mentioned engineered Cas12 proteins or functional derivatives thereof their sequences also include amino acid sequences having the same amino acid sequence as shown in any one of SEQ ID NO: 5, 7, 9 or 12.
  • Amino acid sequences with more than 90% identity may be, for example, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, More than 99.1%, more than 99.2%, more than 99.3%, more than 99.4%, more than 99.5%, more than 99.6%, more than 99.7%, more than 99.8%, more than 99.9%.
  • This application also provides a detection system for detecting target nucleic acid molecules, which system includes: any one or more of the above engineered Cas12 proteins or functional derivatives thereof; guide RNA, which guides the Cas12 protein or Its functional derivatives specifically bind to target nucleic acid molecules; and nucleic acid probes.
  • the target nucleic acid molecule can be target RNA or target DNA.
  • the nucleic acid probe is an RNA probe, and the specific composition and length of the RNA probe can be designed according to actual needs.
  • the sequence of the RNA probe is 5'FAM-rUrUrUrArGrCrArGrGrArUrUrCrArGrGrUrUrCrArGrGrUrUrU-3'BHQ1 (SEQ ID NO: 17).
  • the sequence of the RNA probe is 5'FAM-rCrArUrArUrUrGrArCrGrCrArUrArCrArArUrUrCrCrArCrArGrArGrCrCrU-3'BHQ1 (SEQ ID NO: 18).
  • the sequence of the RNA probe is 5'FAM-rCrCrCrCrCrCrCrCrCrCrCrCrCrCrCrCrCrC-3'BHQ1 (SEQ ID NO: 19).
  • This application also provides a method for detecting target nucleic acid molecules in a sample, including: contacting the sample with any one or more of the above engineered Cas12 proteins or functional derivatives thereof, guide RNA, and target nucleic acid molecules; and measuring through the The engineered Cas12 protein or its functional derivative generates a detectable signal by cleaving the nucleic acid probe, thereby detecting the target nucleic acid molecule.
  • the target nucleic acid molecule can be target RNA or target DNA.
  • the nucleic acid probe is an RNA probe, and the specific composition and length of the RNA probe can be designed according to actual needs.
  • the sequence of the RNA probe is 5'FAM-rUrUrUrArGrCrArGrGrArUrUrCrArGrGrUrUrCrArGrGrUrUrU-3'BHQ1 (SEQ ID NO: 17).
  • the sequence of the RNA probe is 5'FAM-rCrArUrArUrUrGrArCrGrCrArUrArCrArArUrUrCrCrArCrArGrArGrCrCrU-3'BHQ1 (SEQ ID NO: 18).
  • the sequence of the RNA probe is 5'FAM-rCrCrCrCrCrCrCrCrCrCrCrCrCrCrCrCrCrC-3'BHQ1 (SEQ ID NO: 19).
  • This application also provides a detection system for selectively detecting target nucleic acid molecules.
  • the system includes: two or more Cas12 proteins or functional derivatives thereof that have significant differences in the cleavage specificity of RNA and DNA; guide RNA, the Guide RNA guides Cas12 protein or its functional derivatives to specifically bind to target nucleic acid molecules; and nucleic acid probes.
  • the two or more Cas12 proteins or functional derivatives thereof may be two, three, four, or more.
  • At least one Cas12 protein or functional derivative thereof that has a significant difference in the cleavage specificity of RNA and DNA is any of the above-mentioned engineered Cas12 proteins or functional derivatives thereof.
  • the target nucleic acid molecule can be target RNA or target DNA.
  • the nucleic acid probe is a DNA probe or an RNA probe, and the specific composition and length of the DNA probe and RNA probe can be designed according to actual needs.
  • the sequence of the RNA probe is 5'FAM-rUrUrUrArGrCrArGrGrArUrUrCrArGrGrUrUrCrArGrGrUrUrU-3'BHQ1 (SEQ ID NO: 17).
  • the sequence of the RNA probe is 5'FAM-rCrArUrArUrUrGrArCrGrCrArUrArCrArArUrUrCrCrArCrArGrArGrCrCrU-3'BHQ1 (SEQ ID NO: 18).
  • the sequence of the RNA probe is 5'FAM-rCrCrCrCrCrCrCrCrCrCrCrCrCrCrCrCrCrC-3'BHQ1 (SEQ ID NO: 19).
  • the specific domain of the Cas12 protein is a domain that specifically captures nucleic acids and affects the ability to specifically capture nucleic acid trans-cleavage reaction substrates. Therefore, by knocking out this domain, the trans-cleavage reaction activity of either DNA or RNA substrates is greatly weakened. On this basis, by complementing different nucleic acid-specific binding domains, theoretically different nucleic acid capture abilities can be formed and presented. Hand RuvC the ability to do the cutting. In this application, the applicant tried to replace the zinc finger structure of Cas12g, which has higher homology, and Ncp7, which has lower homology in this segment.
  • RNA - Cas12g has been proven to have a much higher ability to cleave RNA than DNA, while Ncp7 is a structural protein that specifically binds RNA. Both substitutions of the original Cas12b domain were confirmed in this application to have higher RNA recognition and cleavage capabilities. Furthermore, after replacing the zinc finger structures recognized by other specific bases, the resulting engineered Cas12 protein can obtain specific recognition and cleavage resolution capabilities for specific base combinations.
  • SEQ ID NO:13 in the figure is:
  • SEQ ID NO:14 is:
  • Example 2 Finger structure mutations upstream and downstream of the ⁇ -helix of the gatekeeping amino acid of Cas12b and protein acquisition.
  • the wild-type AaCas12b was mutated in the following four ways. Among them, the amino acid sequence of wild-type AaCas12b is shown in SEQ ID NO:1, and the amino acid sequence of wild-type Cas12g1 is shown in SEQ ID NO:2.
  • Example 3 Verification of DNA probe cleavage activity of several mutant proteins, using wild-type AaCas12b as a control.
  • each component was placed in a qPCR instrument for fluorescence value analysis.
  • Mutant or wild-type AaCas12b, target sgRNA 150ng, purchased from GenScript Biotechnology Co., Ltd., sequence It is: GTCTAAAGGACAGATTTTCAACGGGTGTGCCAATGGCCACTTTCCAGGTGGCAAAGCCCGTTGAACTTCAAGCGAAGTGGCACACTCAATACTTGAGCACACT (SEQ ID NO: 15)
  • RNase inhibitor ssDNA probe/ssRNA probe (purchased from Shanghai Bailig Biotechnology Co., Ltd.)
  • target synthesis template (1E12 copy, purchased from Shanghai Bailig Biotechnology) Co., Ltd.
  • was placed in 1 ⁇ rCutSmart buffer and a qPCR instrument was used to react at 42°C for 60 minutes. The increase in fluorescence intensity in the first 6 minutes was intercepted for fluorescence analysis.
  • the detection results are shown in Figures 3 and 4. Among them, the probe sequence information and sample sequence information
  • Figure 3 shows the CRISPR cleavage results of DNA probes of wild-type and mutant proteins. The results show that compared with the DNA probe cleavage activity of wild-type Cas12b protein, the DNA probe cleavage activity of AaCas12b-SCas12g protein and Cas12b-NCP7 protein decreased. Preferential cleavage of each base occurs Change, the cleavage preference of the two mutant proteins for the dA probe was significantly reduced.
  • Figure 4 shows the DNA probe CRISPR cutting results of the mutated protein.
  • the results show that compared with the AaCas12b-SCas12g protein and Cas12b-NCP7 protein with the zinc finger structure replaced, the mutant AaCas12b-ggg with a truncated replacement region and the non-zinc finger structure replaced AaCas12b-5MPL has a lower ability to cleave DNA probes.
  • Example 4 Verification of RNA probe cleavage activity of several mutant proteins, using wild-type AaCas12b as a control.
  • Example 3 The cleavage system of Example 3 was used to test the activity of various RNA probes.
  • each component was placed in a qPCR instrument for fluorescence value analysis.
  • /ssRNA probe purchased from Shanghai Bailig Biotechnology Co., Ltd.
  • target synthesis template (1E12 copy, purchased from Shanghai Bailig Biotechnology Co., Ltd.) was placed in 1 ⁇ rCutSmart buffer, and a qPCR instrument was used at 42°C. The reaction was carried out for 60 minutes, and the growth of fluorescence intensity in the first 6 minutes was intercepted for fluorescence analysis. The detection results are shown in Figure 5.
  • the probe sequence information is shown in Table 4.
  • RPA system A purchased from Ampu Future
  • B buffer a buffer
  • RPA upstream and downstream primers each 20pM, purchased from Shanghai Sangon Bioengineering Co., Ltd.
  • actual COVID-19 samples were placed Incubate for 30 minutes at 42°C in a qPCR instrument. Samples are selected to detect the actual extracted COVID-19 samples.
  • the component composition of Example 3 the RPA amplified components and the CRISPR cutting system components are placed in the qPCR instrument for fluorescence value analysis.
  • the detection results are shown in Figure 6.
  • the primer sequence information is shown in Table 5
  • the RPA amplification reaction system is shown in Table 6
  • the CRISPR reaction cleavage system is shown in Table 7.
  • results show that after isothermal amplification, the two proteins AaCas12b-SCas12g and Cas12b-NCP7 use specific sgRNA targeting the new coronavirus as a guide, and the 12b-RNA probe can be used to correctly analyze positive and negative clinical samples of the new coronavirus. distinguish.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Plant Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Peptides Or Proteins (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

提供了一种突变的V型CRISPR酶及其应用。通过蛋白突变理性突变技术，把Cas12a或Cas12b中RuvC识别切割DNA底物的限制去除，使其能够高效率地识别和切割RNA底物，在实际应用中解决RNA底物识别的效率不足的问题。提高Cas12反式切割活性打开后对RNA底物反应效率，提高Cas12使用RNA探针的报告效率。

Description

一种突变的V型CRISPR酶及其应用 技术领域
本申请属于生物技术领域，具体地，涉及一种突变的V型CRISPR酶及其应用。
背景技术
V型CRISPR-Cas系统也被称为Cas12家族，它和其它CRISPR-Cas系统区别之处在于它是由单RuvC活性中心驱动的一个RNA介导的单效应子核酶。随着越来越多的Cas12被挖掘和识别出来，该家族已存在V-A，V-B，V-C，V-D，V-E，V-F，V-G，V-H，V-I，V-J，V-K等多个类别。
其中Cas12a和Cas12b由于其优异的反应性能，被用于多个与扩增技术联合的核酸检测技术及临床检测产品中。
在继往的报道中，Cas12a或Cas12b都是具备RNA介导的顺式DNase活性和反式DNase的活性，以及应运而生的检测应用；在某些报道中虽有提及Cas12a或Cas12b具备RNA介导的反式RNase的活性，可以以比DNA底物1/10的效率进行Rna底物的切割，但其对DNA底物的优先选择性，限制了在核酸扩增体系中其进一步的应用。这种有限的一个数量级的区分度，RNA探针作为另外一种报告底物进行检测的可能性。
发明内容
针对现有技术存在的问题，本申请提供一种突变的V型CRISPR酶及其应用。
具体来说，本申请涉及如下方面：
1.一种工程化的Cas12蛋白或其功能衍生物，其包含如下突变：
将参比Cas12蛋白中RuvC和/或Nuc活性中心识别切割DNA底物的限制去除，
优选地，参比Cas12蛋白的氨基酸序列如SEQ ID NO:1所示。
2.根据项1所述的工程化的Cas12蛋白或其功能衍生物，其中将参比Cas12 蛋白中RuvC和/或Nuc活性中心识别切割DNA底物的限制去除通过以下方式实现：
将参比Cas12蛋白中的守门氨基酸的α螺旋上下游的手指替换为锌指结构域。
3.根据项1或2所述的工程化的Cas12蛋白或其功能衍生物，其中将参比Cas12蛋白中RuvC和/或Nuc活性中心识别切割DNA底物的限制去除是针对915-945位中的部分或全部区域进行结构域的替换，其中氨基酸位置编号如SEQ ID NO:1所定义。
4.根据项2所述的工程化的Cas12蛋白或其功能衍生物，其中所述锌指结构域具有1至2个锌结合位点，所述锌结合位点选自[CxxxxC]、[CxxxxH]、[CxxxC]、[HxxxH]、[CxxC]、[CxxH]中的一种，其中x表示任意天然氨基酸。
5.根据项4所述的工程化的Cas12蛋白或其功能衍生物，其中所述锌指结构域为Cas12g或HIV-1核壳体蛋白7(Ncp7)的锌指结构域。
6.根据项5所述的工程化的Cas12蛋白或其功能衍生物，其中参比Cas12蛋白的919-944位替换为SEQ ID NO:4所示的氨基酸序列，其中氨基酸位置编号如SEQ ID NO:1所定义。
7.根据项5所述的工程化的Cas12蛋白或其功能衍生物，其中参比Cas12蛋白的919-944位替换为SEQ ID NO:6所示的氨基酸序列，其中氨基酸位置编号如SEQ ID NO:1所定义。
8.根据项1所述的工程化的Cas12蛋白或其功能衍生物，其中参比Cas12蛋白的919-944位替换为SEQ ID NO:8所示的氨基酸序列，其中氨基酸位置编号如SEQ ID NO:1所定义。
9.根据项1所述的工程化的Cas12蛋白或其功能衍生物，其中参比Cas12蛋白的916-944位替换为SEQ ID NO:11所示的氨基酸序列，其中氨基酸位置编号如SEQ ID NO:1所定义。
10.一种工程化的Cas12蛋白或其功能衍生物，其包含基于参比Cas12蛋白的如下突变中的一种：
参比Cas12蛋白的919-944位替换为SEQ ID NO:4所示的氨基酸序列；
参比Cas12蛋白的919-944位替换为SEQ ID NO:6所示的氨基酸序列；
参比Cas12蛋白的919-944位替换为SEQ ID NO:8所示的氨基酸序列；
参比Cas12蛋白的916-944位替换为SEQ ID NO:11所示的氨基酸序列；
其中参比Cas12蛋白的氨基酸序列如SEQ ID NO:1所示。
11.一种工程化的Cas12蛋白或其功能衍生物，其序列为如SEQ ID NO:5、7、9或12中任一项所示的氨基酸序列；或与如SEQ ID NO:5、7、9或12中任一项所示的氨基酸序列具有90％以上同一性的氨基酸序列。
12.一种用于检测靶标核酸分子的检测体系，所述体系包含：
项1-11中任一项所述的工程化的Cas12蛋白或其功能衍生物；
向导RNA，所述向导RNA引导Cas12蛋白或其功能衍生物特异性结合于靶标核酸分子；和核酸探针。
13.根据项12所述的检测体系，其中所述核酸探针为RNA探针。
14.一种检测样品中靶标核酸分子的方法，包括：
使样品与项1-11中任一项所述的工程化的Cas12蛋白或其功能衍生物、向导RNA和靶标核酸分子接触；以及
测量通过所述工程化的Cas12蛋白或其功能衍生物切割核酸探针而产生的可检测信号，从而检测所述靶标核酸分子。
15.根据项14所述的方法，其中核酸探针为RNA探针。
16.一种选择性检测靶标核酸分子的检测体系，所述体系包含：
两种以上对于RNA和DNA的切割特异性有显著性差异的Cas12蛋白或其功能衍生物；
向导RNA，所述向导RNA引导Cas12蛋白或其功能衍生物特异性结合于靶标核酸分子；和
核酸探针。
17.根据项16所述的检测体系，其中至少一种所述Cas12蛋白或其功能衍生物为项1-11中任一项所述的工程化的Cas12蛋白或其功能衍生物。
18.根据项17所述的检测体系，其中所述核酸探针为DNA探针或RNA探针。
本申请通过蛋白突变理性突变技术，把Cas12b中RuvC识别切割DNA底物的限制去除，可以特异性的识别DNA底物或特异性的识别RNA底物，在实际应用中解决DNA/RNA底物识别的区分度不足的问题。
本申请的工程化的Cas12蛋白或其功能衍生物提高Cas12反式切割活性打开 后对RNA底物反应效率，提高Cas12使用RNA探针的报告效率。
附图说明
图1显示Cas12b和Cas12g的序列比对结果；
图2显示4种突变蛋白的电泳图；
图3显示野生型和突变蛋白的DNA探针CRISPR切割结果；
图4显示突变蛋白的DNA探针CRISPR切割结果；
图5显示突变蛋白的RNA探针CRISPR切割结果；
图6显示突变蛋白应用于RPA扩增反应体系CRISPR切割结果。
具体实施方式
下面结合实施例进一步说明本申请，应当理解，实施例仅用于进一步说明和阐释本申请，并非用于限制本申请。
除非另外定义，本说明书中有关技术的和科学的术语与本领域内的技术人员所通常理解的意思相同。虽然在实验或实际应用中可以应用与此间所述相似或相同的方法和材料，本文还是在下文中对材料和方法做了描述。在相冲突的情况下，以本说明书包括其中定义为准，另外，材料、方法和例子仅供说明，而不具限制性。以下结合具体实施例对本申请作进一步的说明，但不用来限制本申请的范围。
定义
如本文所用，术语“Cas12”或“Cas12蛋白”包括Cas12a(也称为Cpf1)、Cas12b、Cas12c、Cas12d、Cas12e、Cas12h、Cas12i、Cas12g等。在一些实施方案中，Cas12蛋白是Cas12b蛋白，Cas12b蛋白在其最广泛的意义上使用，并且包括亲本或参考Cas12b蛋白质(例如，氨基酸序列为SEQ ID NO:1的AaCas12b)、其衍生物或变体以及功能性片段，诸如其寡核苷酸结合片段。
如本文所用，某种蛋白的“功能衍生物”包括所述蛋白的各种变体或功能结构域，只要所述变体或功能结构域保留了所述蛋白的某个功能结构域的功能(无论是增强的所述功能或减弱的所述功能)，即可称为所述蛋白的功能衍生物。例如对于Cas12蛋白，保留了其部分结构域功能的Cas12蛋白变体或截短体均属于Cas12蛋白的功能衍生物。
如本文所用，“结构域”或“蛋白质结构域”是指可以独立于该蛋白质链的其余部分而存在并且起作用的蛋白质序列的一部分。
如本文所用，“向导RNA”、“sgRNA”“gRNA”在本文中可互换使用，是指能够与Cas12蛋白和靶标核酸形成复合物的RNA。
如本文所用，术语“核酸”、“多核苷酸”和“核苷酸序列”可互换使用，是指任何长度的核苷酸的聚合形式，包括脱氧核糖核苷酸、核糖核苷酸、其组合及其类似物。“寡核苷酸”和“低聚核苷酸”可互换使用，是指具有不超过约50个核苷酸的短多核苷酸。
如本文所用，核酸的“互补”是指一条核酸通过传统的Watson-Crick碱基配对与另一条核酸形成氢键的能力。百分比互补性表示核酸分子中可与另一核酸分子形成氢键(即，Watson-Crick碱基配对)的残基的百分比(例如，10个中的约5、6、7、8、9、10个分别为约50％，60％，70％，80％，90％和100％互补)。“完全互补”是指核酸序列的所有连续残基与第二核酸序列中相同数量的连续残基形成氢键。如本文所用，“基本上互补”是指在约40、50、60、70、80、100、150、200、250或更多个核苷酸的区域内，至少约70％，75％，80％，85％，90％，95％，97％，98％，99％或100％中的任何一个的互补程度，或指在严格条件下杂交的两条核酸。对于单个碱基或单个核苷酸，按照Watson-Crick碱基配对原则，A与T或U、C与G或I配对时，被称为互补或匹配，反之亦然；而除此以外的碱基配对都称为不互补或不匹配。如非特别指出，本申请的“互补”包含“完全互补”和“基本上互补”的情况。只要两条核酸序列可以通过Walson-Crick碱基配对形成稳定的杂合双链，则称所述两条核酸序列“互补”，其形成稳定的杂合双链的过程称为“互补杂交”。
如本文所用，术语“野生型”具有本领域技术人员通常理解的含义，意指当它存在于大自然中时，将其与突变体或变体区分开的、典型形式的生物体、菌株、基因或特征。它可以与自然界中的资源隔离开来，并没有被刻意修饰。
如本文所用，术语“非天然存在”或“工程化的”可互换使用，是指人工参与。当这些术语用于描述核酸分子或多肽时，是指所述核酸分子或多肽至少基本上不含其天然缔合的或天然存在的至少一种其他组分。
如本文所用，术语“同一性”用于表示两个多肽之间或两个核酸之间的序列匹配。当两个比较序列中的一个位置被相同的碱基或氨基酸单体亚基占据时 (例如，两个DNA分子的每个中的一个位置都被腺嘌呤占据，或者两个多肽的每个中的一个位置被赖氨酸占据)，那么在那个位置每个分子均相同。这两个序列之间的“同一性百分比”是两个序列共有的匹配位置数除以要比较的位置数x 100的函数。例如，如果两个序列的10个位置中有6个匹配，则这两个序列具有60％的同一性。例如，DNA序列CTGACT和CAGGTT具有50％的同一性(总共6个位置中有3个匹配)。通常，当两个序列进行比对以产生最大的同一性时，进行这种比较。这种比对可以通过例如Needleman et al.(1970)J.Mol.Biol.48:443-453中的方法来实现，所述方法可方便地通过计算机程序如比对(Align)程序(DNAstar，Inc.)来进行。也可以采用PAM 120权重残基表，使用E.Meyers和W.Miller的算法(Comput.Appl Biosci.,4:11-17(1988))集成到ALIGN程序(2.0版)中。空缺长度罚分12和空缺罚分4，用于确定两个氨基酸序列之间的同一性百分比。此外，可以使用集成到GCG软件包(可从www.gcg.com获得)的GAP程序中的Needleman和Wunsch(J MoI Biol.48:444-453(1970))算法，采用Blossum 62矩阵或PAM250矩阵，空缺权重为16、14、12、10、8、6或4，长度权重为1、2、3、4、5或6，以确定两个氨基酸序列之间的同一性百分比。
如本文所用，“变体”或“突变体”解释为分别不同于参比多核苷酸或多肽但保留必要特性的多核苷酸或多肽。多核苷酸的典型变体与另一参比多核苷酸的核酸序列不同。变体核酸序列的变化可以改变或可以不改变参比多核苷酸编码的多肽的氨基酸序列。核苷酸变化可导致参比序列编码的多肽中的氨基酸替换、添加、缺失、融合和截短，如下所述。多肽的典型变体与另一参比多肽在氨基酸序列上不同。通常，差异是有限的，使得参比多肽和变体的序列总体上非常相似，并且在许多区域是相同的。变体和参比多肽的氨基酸序列可以通过一个或多个替换、添加、缺失的任何组合而不同。替换或插入的氨基酸残基可以是或可以不是遗传密码编码的氨基酸残基。多核苷酸或多肽的变体可以是天然存在的(诸如等位基因变体)，或者可以是未知天然存在的变体。多核苷酸和多肽的非天然存在的变体可以通过诱变技术，通过直接合成，以及通过本领域技术人员已知的其他重组方法来制备。
如本文所用，术语“靶标核酸”或“靶标核酸分子”是指样品中的目标核酸，可以是靶标RNA，也可以是靶标DNA，还可以同时含有靶标RNA和靶标 DNA。
本申请提供一种工程化的Cas12蛋白或其功能衍生物，其包含如下突变：
将参比Cas12蛋白中RuvC和/或Nuc活性中心识别切割DNA底物的特异性进行改造。其中，参比Cas12蛋白可以是已知的野生型Cas12蛋白。在一些实施方案中，参比Cas12蛋白可以是已知的野生型Cas12蛋白。在一些实施方案中，参比Cas12蛋白可以是野生型Cas12b蛋白。
在一些实施方案中，参比Cas12蛋白可以是野生型AaCas12b蛋白。其氨基酸序列如SEQ ID NO:1所示。
在一些实施方案中，将参比Cas12蛋白中RuvC活性中心识别切割DNA底物的特异性进行改造。
在一些实施方案中，将参比Cas12蛋白中Nuc活性中心识别切割DNA底物的特异性进行改造。
在一些实施方案中，将参比Cas12蛋白中RuvC和Nuc活性中心识别切割DNA底物的特异性进行改造。
在一些实施方案中，将参比Cas12蛋白中RuvC和/或Nuc活性中心识别切割DNA底物的特异性进行改造包括如下方式：
将参比Cas12蛋白中的守门氨基酸的α螺旋上下游的手指替换为锌指结构域。
在一些实施方案中，将参比Cas12蛋白中RuvC和/或Nuc活性中心识别切割DNA底物的限制去除是针对915-945位中的部分或全部区域进行结构域的替换，其中氨基酸位置编号如SEQ ID NO:1所定义。其中的部分区域可以是915-945位中的一个位点或多个位点，也可以是一段区域多段区域。例如，部分区域可以是位点P916，或位点L941，或位点P916和位点L941；部分区域可以是919-944位，或916-944位。
在一些实施方案中，将参比Cas12蛋白中RuvC和/或Nuc活性中心识别切割DNA底物的限制去除是针对919-944位中的部分或全部区域进行结构域的替换，其中氨基酸位置编号如SEQ ID NO:1所定义。
在一些实施方案中，将参比Cas12蛋白中RuvC和/或Nuc活性中心识别切割DNA底物的限制去除是针对916-944位中的部分或全部区域进行结构域的替换，例如替换为锌指结构域，其中氨基酸位置编号如SEQ ID NO:1所定义。
在一些实施方案中，所述锌指结构域具有1至2个锌结合位点，所述锌结合位点选自[CxxxxC]、[CxxxxH]、[CxxxC]、[HxxxH]、[CxxC]、[CxxH]中的一种，其中x表示任意天然氨基酸。
在一些实施方案中，所述锌指结构域为Cas12g的锌指结构域。
在一些实施方案中，所述锌指结构域为HIV-1核壳体蛋白7(Ncp7)的锌指结构域。
在一些实施方案中，参比Cas12蛋白的919-944位替换为Cas12g的锌指结构域。
在一些实施方案中，参比Cas12蛋白的919-944位替换为SEQ ID NO:4所示的氨基酸序列，其中氨基酸位置编号如SEQ ID NO:1所定义。得到的工程化Cas12蛋白的氨基酸序列如SEQ ID NO:5所示。
在一些实施方案中，参比Cas12蛋白的919-944位替换为Ncp7的锌指结构域。
在一些实施方案中，参比Cas12蛋白的919-944位替换为SEQ ID NO:6所示的氨基酸序列，其中氨基酸位置编号如SEQ ID NO:1所定义。得到的工程化Cas12蛋白的氨基酸序列如SEQ ID NO:7所示。
在一些实施方案中，对参比Cas12蛋白中RuvC和/或Nuc活性中心识别切割DNA底物的特异性进行改造是通过将参比Cas12蛋白中的守门氨基酸的919-944位替换为SEQ ID NO:8所示的氨基酸序列，其中氨基酸位置编号如SEQ ID NO:1所定义。得到的工程化Cas12蛋白的氨基酸序列如SEQ ID NO:9所示。
在一些实施方案中，对参比Cas12蛋白中RuvC和/或Nuc活性中心识别切割DNA底物的特异性进行改造是通过将参比Cas12蛋白中的守门氨基酸的916-944位替换为SEQ ID NO:11所示的氨基酸序列，其中氨基酸位置编号如SEQ ID NO:1所定义。得到的工程化Cas12蛋白的氨基酸序列如SEQ ID NO:12所示。
本申请还提供一种工程化的Cas12蛋白或其功能衍生物，其包含基于参比Cas12蛋白的如下突变中的一种：
参比Cas12蛋白的919-944位替换为SEQ ID NO:4所示的氨基酸序列；
参比Cas12蛋白的919-944位替换为SEQ ID NO:6所示的氨基酸序列；
参比Cas12蛋白的919-944位替换为SEQ ID NO:8所示的氨基酸序列；
参比Cas12蛋白的916-944位替换为SEQ ID NO:11所示的氨基酸序列；
其中参比Cas12蛋白的氨基酸序列如SEQ ID NO:1所示。
本领域技术人员可以理解，对于上述任意一种工程化的Cas12蛋白或其功能衍生物，其序列还涵盖与如SEQ ID NO:5、7、9或12中任一项所示的氨基酸序列具有90％以上同一性的氨基酸序列。其中90％以上同一性，可以例如为90％以上、91％以上、92％以上、93％以上、94％以上、95％以上、96％以上、97％以上、98％以上、99％以上、99.1％以上、99.2％以上、99.3％以上、99.4％以上、99.5％以上、99.6％以上、99.7％以上、99.8％以上、99.9％以上。
本申请还提供一种用于检测靶标核酸分子的检测体系，所述体系包含：上述任意一种或多种工程化的Cas12蛋白或其功能衍生物；向导RNA，所述向导RNA引导Cas12蛋白或其功能衍生物特异性结合于靶标核酸分子；和核酸探针。其中，靶标核酸分子可以为靶标RNA，也可以为靶标DNA。
在一些实施方案中，核酸探针为RNA探针，RNA探针的具体组成和长度可以根据实际需要进行设计。
在一些实施方案中，RNA探针的序列为5‘FAM-rUrUrUrArGrCrArGrGrArUrUrCrArGrGrUrUrU-3’BHQ1(SEQ ID NO:17)。
在一些实施方案中，RNA探针的序列为5‘FAM-rCrArUrArUrUrGrArCrGrCrArUrArCrArArArArCrArUrUrCrCrCrArCrCrArArCrArGrArGrCrCrU-3’BHQ1(SEQ ID NO:18)。
在一些实施方案中，RNA探针的序列为5‘FAM-rCrCrCrCrCrCrCrCrCrC-3’BHQ1(SEQ ID NO:19)。
本申请还提供一种检测样品中靶标核酸分子的方法，包括：使样品与上述任意一种或多种工程化的Cas12蛋白或其功能衍生物、向导RNA和靶标核酸分子接触；以及测量通过所述工程化的Cas12蛋白或其功能衍生物切割核酸探针而产生的可检测信号，从而检测所述靶标核酸分子。其中，靶标核酸分子可以为靶标RNA，也可以为靶标DNA。
在一些实施方案中，核酸探针为RNA探针，RNA探针的具体组成和长度可以根据实际需要进行设计。
在一些实施方案中，RNA探针的序列为5‘FAM-rUrUrUrArGrCrArGrGrArUrUrCrArGrGrUrUrU-3’BHQ1(SEQ ID NO:17)。
在一些实施方案中，RNA探针的序列为5‘FAM-rCrArUrArUrUrGrArCrGrCrArUrArCrArArArArCrArUrUrCrCrCrArCrCrArArCrArGrArGrCrCrU-3’BHQ1(SEQ ID NO:18)。
在一些实施方案中，RNA探针的序列为5‘FAM-rCrCrCrCrCrCrCrCrCrC-3’BHQ1(SEQ ID NO:19)。
本申请还提供一种选择性检测靶标核酸分子的检测体系，所述体系包含：两种以上对于RNA和DNA的切割特异性有显著性差异的Cas12蛋白或其功能衍生物；向导RNA，所述向导RNA引导Cas12蛋白或其功能衍生物特异性结合于靶标核酸分子；和核酸探针。其中，两种以上的Cas12蛋白或其功能衍生物可以为两种、三种、四种、或更多种。
在一些实施方案中，至少一种对于RNA和DNA的切割特异性有显著性差异的Cas12蛋白或其功能衍生物为上述任意一种工程化的Cas12蛋白或其功能衍生物。其中，靶标核酸分子可以为靶标RNA，也可以为靶标DNA。
在一些实施方案中，核酸探针为DNA探针或RNA探针，DNA探针和RNA探针的具体组成和长度可以根据实际需要进行设计。
在一些实施方案中，RNA探针的序列为5‘FAM-rUrUrUrArGrCrArGrGrArUrUrCrArGrGrUrUrU-3’BHQ1(SEQ ID NO:17)。
在一些实施方案中，RNA探针的序列为5‘FAM-rCrArUrArUrUrGrArCrGrCrArUrArCrArArArArCrArUrUrCrCrCrArCrCrArArCrArGrArGrCrCrU-3’BHQ1(SEQ ID NO:18)。
在一些实施方案中，RNA探针的序列为5‘FAM-rCrCrCrCrCrCrCrCrCrC-3’BHQ1(SEQ ID NO:19)。
本申请发现Cas12蛋白的特定结构域是核酸特异性俘获的结构域，影响核酸反式切割反应底物的特异性俘获的能力。因此，通过敲除该结构域，得到无论是DNA或者RNA底物反式切割反应活力大大削弱的结果。在此基础上通过回补不同的核酸特异性结合的结构域，理论上可以形成不同核酸俘获能力并呈 递给RuvC进行切割的能力。在本申请中，申请人尝试置换了该区段同源性较高的Cas12g，和同源性较低的Ncp7的锌指结构。两个结构域均为对RNA有偏好性识别的结构域——Cas12g已经被证明了对RNA的切割能力远远高于对DNA的切割能力，而Ncp7则是RNA特异性结合的结构蛋白，这两者对原有Cas12b结构域的置换均在本申请中证实具有更高的RNA识别和切割能力。进一步地，置换其他特异性碱基识别的锌指结构后，得到的工程化的Cas12蛋白能获得具体碱基组合特异性识别以及切割的分辨能力。
实施例
实施例1——Cas12b和Cas12g的序列比对和偏好性分析
使用CLUSTAL 2.1 Mμltiple Sequence Alignments工具，对Cas12b和Cas12g进行同源比对，结果如图1所示。
图中SEQ ID NO:13为：
SEQ ID NO:14为：
发现二者在RuvC(Nuv)区域的相似性比较高。识别出了Cas12b中与RuvC配合的α螺旋的与Cas12g1同源的结构，以及α螺旋上下游潜在的可形成更强锌指的位点P916和L941。
实施例2——Cas12b的守门氨基酸的α螺旋上下游的手指结构突变及蛋白质获取。
对野生型AaCas12b进行以下4种方式的突变。其中，野生型AaCas12b的氨基酸序列如SEQ ID NO:1所示，野生型Cas12g1的氨基酸序列如SEQ ID NO:2所示。
SEQ ID NO:1

SEQ ID NO:2
(1)将野生型AaCas12b的氨基酸序列的919-944区段(该区段的氨基酸序列为CAREQNPEPFPWWLNKFVAEHKLDGC(SEQ ID NO:3))，替换为Cas12g1的同源区段(该区段的氨基酸序列为CARCRKKQKDNKQWEKNKKRGLFKCEGC(SEQ ID NO:4))，得到的蛋白称为AaCas12b-SCas12g，其氨基酸序列如SEQ ID NO:5所示。其中下划线部分表示替换的区段。
SEQ ID NO:5

(2)将野生型AaCas12b的氨基酸序列的919-944区段，替换为Ncp7锌指区段(该区段的氨基酸序列为CFNCGKEGHTARNCRAPRKKGCWKCGKEGHNMKDC(SEQ ID NO:6))，得到的蛋白称为AaCas12b-NCP7，其氨基酸序列如SEQ ID NO:7所示。其中下划线部分表示替换的区段。
SEQ ID NO:7
(3)将野生型AaCas12b的氨基酸序列的919-944区段，替换为5MPL蛋白的区段(该区段的氨基酸序列为SQRPGAHLTVKKIFVGGIKEDTEEHHLRDYFEQYGKIEVIEIMTDRGSGKKRGFAFVTFDDHDSVDKIVIQKYHTVNGHNCEVRKALSKQEMASASSSQRGR(SEQ ID NO:8))，得到的蛋白称为AaCas12b-5MPL，其氨基酸序列如SEQ ID NO:9所示。其中下划线部分表示替换的区段。
SEQ ID NO:9

(4)将野生型AaCas12b的氨基酸序列的916-944区段(该区段的氨基酸序列为CAREQNPEPFPWWLNKFVAEHKLDGCPLR(SEQ ID NO:10))，替换为GGGGGG(SEQ ID NO:11)得到的蛋白称为AaCas12b-ggg，其氨基酸序列如SEQ ID NO:12所示。其中下划线部分表示替换的区段。
SEQ ID NO:12
根据以上4种突变体的氨基酸序列设计相应的载体，转入pET28表达载体中 在大肠杆菌菌株BL21中进行诱导表达，通过亲和层析、离子交换层析等蛋白纯化方法，得到4种相应的突变蛋白。图2为纯化好的4种蛋白的聚丙烯酰胺凝胶电泳图。
实施例3——几种突变蛋白的DNA探针切割活性验证，以野生型的AaCas12b为对照。
具体地，按照表1所示的组分构成将各组分置于qPCR仪器中进行荧光值分析，将突变型或野生型AaCas12b，靶标sgRNA(150ng，购自金斯瑞生物科技有限公司，序列为：GTCTAAAGGACAGATTTTCAACGGGTGTGCCAATGGCCACTTTCCAGGTGGCAAAGCCCGTTGAACTTCAAGCGAAGTGGCACACTCAATACTTGAGCACACT(SEQ ID NO:15))，RNase inhibitor，ssDNA探针/ssRNA探针(购自上海百力格生物技术有限公司)，靶标合成模板(1E12拷贝，购自上海百力格生物技术有限公司)置于1×rCutSmart缓冲液中，使用qPCR仪在42℃下反应60min，截取前6分钟的荧光强度增长进行荧光分析，检测结果如图3和图4所示。其中，探针序列信息和样本序列信息如表2和表3所示。
表1新冠O靶的Crispr切割体系
表2探针序列信息

表3样本序列信息
其中图3为野生型和突变蛋白的DNA探针CRISPR切割结果，结果显示，对比野生型Cas12b蛋白的DNA探针切割活力，AaCas12b-SCas12g蛋白和Cas12b-NCP7蛋白对DNA探针的切割活力下降，对各碱基切割的偏好性发生 改变，两个突变体蛋白对dA探针的切割偏好性明显下降。
图4为突变蛋白的DNA探针CRISPR切割结果，结果显示，对比置换了锌指结构的AaCas12b-SCas12g蛋白和Cas12b-NCP7蛋白，置换区域截短的突变AaCas12b-ggg和置换了非锌指结构的AaCas12b-5MPL对DNA探针的切割能力更低。
实施例4—几种突变蛋白的RNA探针切割活性验证，以野生型的AaCas12b为对照。
使用实施例3的切割体系测试各种RNA探针的活性。
具体地，按照实施例3的组分构成将各组分置于qPCR仪器中进行荧光值分析，我们将突变型或野生型AaCas12b，靶标sgRNA(SEQ ID NO:15)，RNase inhibitor，ssDNA探针/ssRNA探针(购自上海百力格生物技术有限公司)，靶标合成模板(1E12拷贝，购自上海百力格生物技术有限公司)置于1×rCutSmart缓冲液中，使用qPCR仪在42℃下反应60min，截取前6分钟的荧光强度增长进行荧光分析，检测结果如图5所示。其中探针序列信息如表4所示。
表4探针序列信息
结果显示，野生型Cas12b对不同长度的RNA探针切割活力相近。对于12b-RNA probe探针的反应上，突变后的AaCas12b-SCas12g和Cas12b-NCP7蛋白切割活力要比野生型Cas12b蛋白切割各种RNA探针要高。
实施例5—AaCas12b-SCas12g和Cas12b-NCP7应用RNA探针进行检测的应用
具体地，按照试剂盒(购买自安普未来)构成将RPA体系A,B buffer，RPA上下游引物(各20pM，购自上海生工生物工程有限公司)，新冠实际样本置于 qPCR仪器中42℃孵育30min，样本选择检测真实提取的新冠样本，按照实施例3的组分构成将RPA扩增后的组分与CRISPR切割体系组分置于qPCR仪器中进行荧光值分析，将突变型或野生型AaCas12b，靶标sgRNA(SEQ ID NO:15)，RNase inhibitor，ssRNA探针(购自上海百力格生物技术有限公司)，置于1×rCutSmart缓冲液中，使用qPCR仪在42℃下反应60min，截取前6分钟的荧光强度增长进行荧光分析，检测结果如图6所示。其中，引物序列信息如表5所示，RPA扩增反应体系如表6所示，CRISPR反应切割体系如表7所示。
表5 RPA引物序列信息
表6 RPA扩增反应体系
表7 CRISPR反应切割体系

结果显示，经过等温扩增后，AaCas12b-SCas12g和Cas12b-NCP7两个蛋白使用靶向新冠病毒的特异性sgRNA作为引导，应用12b-RNA probe探针，能对阳性和阴性的新冠临床样本进行正确区分。

Claims (18)

  1. 一种工程化的Cas12蛋白或其功能衍生物，其包含如下突变：
    将参比Cas12蛋白中RuvC和/或Nuc活性中心识别切割DNA底物的限制去除，
    优选地，参比Cas12蛋白的氨基酸序列如SEQ ID NO:1所示。
  2. 根据权利要求1所述的工程化的Cas12蛋白或其功能衍生物，其中将参比Cas12蛋白中RuvC和/或Nuc活性中心识别切割DNA底物的限制去除通过如下方式实现：
    将参比Cas12蛋白中的守门氨基酸的α螺旋上下游的手指替换为锌指结构域。
  3. 根据权利要求1或2所述的工程化的Cas12蛋白或其功能衍生物，其中将参比Cas12蛋白中RuvC和/或Nuc活性中心识别切割DNA底物的限制去除是针对915-945位中的部分或全部区域进行结构域的替换，其中氨基酸位置编号如SEQ ID NO:1所定义。
  4. 根据权利要求2所述的工程化的Cas12蛋白或其功能衍生物，其中所述锌指结构域具有1至2个锌结合位点，所述锌结合位点选自[CxxxxC]、[CxxxxH]、[CxxxC]、[HxxxH]、[CxxC]、[CxxH]中的一种，其中x表示任意天然氨基酸。
  5. 根据权利要求4所述的工程化的Cas12蛋白或其功能衍生物，其中所述锌指结构域为Cas12g或HIV-1核壳体蛋白7(Ncp7)的锌指结构域。
  6. 根据权利要求5所述的工程化的Cas12蛋白或其功能衍生物，其中参比Cas12蛋白的919-944位替换为SEQ ID NO:4所示的氨基酸序列，其中氨基酸位置编号如SEQ ID NO:1所定义。
  7. 根据权利要求5所述的工程化的Cas12蛋白或其功能衍生物，其中参比Cas12蛋白的919-944位替换为SEQ ID NO:6所示的氨基酸序列，其中氨基酸位置编号如SEQ ID NO:1所定义。
  8. 根据权利要求1所述的工程化的Cas12蛋白或其功能衍生物，其中参比Cas12蛋白的919-944位替换为SEQ ID NO:8所示的氨基酸序列，其中氨基酸位置编号如SEQ ID NO:1所定义。
  9. 根据权利要求1所述的工程化的Cas12蛋白或其功能衍生物，其中参比 Cas12蛋白的916-944位替换为SEQ ID NO:11所示的氨基酸序列，其中氨基酸位置编号如SEQ ID NO:1所定义。
  10. 一种工程化的Cas12蛋白或其功能衍生物，其包含基于参比Cas12蛋白的如下突变中的一种：
    参比Cas12蛋白的919-944位替换为SEQ ID NO:4所示的氨基酸序列；
    参比Cas12蛋白的919-944位替换为SEQ ID NO:6所示的氨基酸序列；
    参比Cas12蛋白的919-944位替换为SEQ ID NO:8所示的氨基酸序列；
    参比Cas12蛋白的916-944位替换为SEQ ID NO:11所示的氨基酸序列；
    其中参比Cas12蛋白的氨基酸序列如SEQ ID NO:1所示。
  11. 一种工程化的Cas12蛋白或其功能衍生物，其序列为如SEQ ID NO:5、7、9或12中任一项所示的氨基酸序列；或与如SEQ ID NO:5、7、9或12中任一项所示的氨基酸序列具有90％以上同一性的氨基酸序列。
  12. 一种用于检测靶标核酸分子的检测体系，所述体系包含：
    权利要求1-11中任一项所述的工程化的Cas12蛋白或其功能衍生物；
    向导RNA，所述向导RNA引导Cas12蛋白或其功能衍生物特异性结合于靶标核酸分子；
    和核酸探针。
  13. 根据权利要求12所述的检测体系，其中所述核酸探针为RNA探针。
  14. 一种检测样品中靶标核酸分子的方法，包括：
    使样品与权利要求1-11中任一项所述的工程化的Cas12蛋白或其功能衍生物、向导RNA和靶标核酸分子接触；以及
    测量通过所述工程化的Cas12蛋白或其功能衍生物切割核酸探针而产生的可检测信号，从而检测所述靶标核酸分子。
  15. 根据权利要求14所述的方法，其中所述核酸探针为RNA探针。
  16. 一种选择性检测靶标核酸分子的检测体系，所述体系包含：
    两种以上对于RNA和DNA的切割特异性有显著性差异的Cas12蛋白或其功能衍生物；
    向导RNA，所述向导RNA引导Cas12蛋白或其功能衍生物特异性结合于靶标核酸分子；和
    核酸探针。
  17. 根据权利要求16所述的检测体系，其中至少一种所述Cas12蛋白或其功能衍生物为权利要求1-11中任一项所述的工程化的Cas12蛋白或其功能衍生物。
  18. 根据权利要求17所述的检测体系，其中所述核酸探针为DNA探针或RNA探针。
PCT/CN2023/115471 2022-08-29 2023-08-29 一种突变的v型crispr酶及其应用 WO2024046307A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202211039436 2022-08-29
CN202211039436.2 2022-08-29

Publications (1)

Publication Number Publication Date
WO2024046307A1 true WO2024046307A1 (zh) 2024-03-07

Family

ID=90027649

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/115471 WO2024046307A1 (zh) 2022-08-29 2023-08-29 一种突变的v型crispr酶及其应用

Country Status (2)

Country Link
CN (1) CN117625577A (zh)
WO (1) WO2024046307A1 (zh)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109837328A (zh) * 2018-09-20 2019-06-04 中国科学院动物研究所 核酸检测方法
US20200332275A1 (en) * 2018-09-13 2020-10-22 The Board Of Regents Of The University Of Oklahoma Variant cas12 proteins with improved dna cleavage selectivity and methods of use
CN112195164A (zh) * 2020-12-07 2021-01-08 中国科学院动物研究所 工程化的Cas效应蛋白及其使用方法
CN113227367A (zh) * 2018-08-09 2021-08-06 G+Flas生命科学公司 用cas12a蛋白进行基因组工程的组合物和方法
CN113286884A (zh) * 2018-08-07 2021-08-20 博德研究所 新型cas12b酶和系统

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113286884A (zh) * 2018-08-07 2021-08-20 博德研究所 新型cas12b酶和系统
CN113227367A (zh) * 2018-08-09 2021-08-06 G+Flas生命科学公司 用cas12a蛋白进行基因组工程的组合物和方法
US20200332275A1 (en) * 2018-09-13 2020-10-22 The Board Of Regents Of The University Of Oklahoma Variant cas12 proteins with improved dna cleavage selectivity and methods of use
CN109837328A (zh) * 2018-09-20 2019-06-04 中国科学院动物研究所 核酸检测方法
CN112195164A (zh) * 2020-12-07 2021-01-08 中国科学院动物研究所 工程化的Cas效应蛋白及其使用方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHEN XU, TAN YAN, WANG SHUOSHI, WU XUELI, LIU RUI, YANG XINGGUI, WANG YI, TAI JUN, LI SHIJUN: "A CRISPR-Cas12b–Based Platform for Ultrasensitive, Rapid, and Highly Specific Detection of Hepatitis B Virus Genotypes B and C in Clinical Application", FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY, FRONTIERS RESEARCH FOUNDATION, CH, vol. 9, 7 October 2021 (2021-10-07), CH , pages 743322, XP093143041, ISSN: 2296-4185, DOI: 10.3389/fbioe.2021.743322 *
YIXIANG ZHANG, ZHANG QINGPING; LI LINXIAN; WANG LIANG; YANG JIELIN: "Detection of Cronobacter spp in Milk Powder by CRISPR/Cas12b", JOURNAL OF CHINESE INSTITUTE OF FOOD SCIENCE AND TECHNOLOGY, CHINESE INSTITUTE OF FOOD SCIENCE AND TECHNOLOGY, CN, vol. 22, no. 6, 16 June 2022 (2022-06-16), CN , pages 267 - 275, XP093143046, ISSN: 1009-7848, DOI: 10.16429/j.1009-7848.2022.06.028 *

Also Published As

Publication number Publication date
CN117625577A (zh) 2024-03-01

Similar Documents

Publication Publication Date Title
CN110959040A (zh) 具有改进的精度和特异性的碱基编辑器
CN101228268A (zh) Rna扩增和/或rna标记用的rna依赖的rna聚合酶，方法及试剂盒
JP2003510052A (ja) 改良されたポリヌクレオチド合成のための方法と組成物
CN114375341A (zh) 一种检测非洲猪瘟病毒的方法和试剂盒
CN113046475B (zh) 一种快速检测突变型新型冠状病毒的引物组合物及试剂盒
KR102377213B1 (ko) 해양 dna 폴리머라제
CN112639089A (zh) 重组型kod聚合酶
Shi et al. DNA topology regulates PAM-Cas9 interaction and DNA unwinding to enable near-PAMless cleavage by thermophilic Cas9
Acharya et al. Biochemical properties of single-stranded DNA-binding protein from Mycobacterium smegmatis, a fast-growing mycobacterium and its physical and functional interaction with uracil DNA glycosylases
CN112899253B (zh) 具有dna聚合酶活性的多肽、重组载体及其制备方法与应用
WO2024046307A1 (zh) 一种突变的v型crispr酶及其应用
CN112725299B (zh) 改善Taq DNA聚合酶耐受性的突变体及制备方法和应用
CN116410955B (zh) 两种新型核酸内切酶及其在核酸检测中的应用
CN115851714A (zh) 一种基于crispr技术检测甘薯褪绿矮化病毒的方法
CN116376905A (zh) 一种基于crispr技术检测甘薯褪绿斑病毒的方法
CN116103286A (zh) 一种基于crispr技术检测甘薯羽状斑驳病毒的方法
CN116042569A (zh) Mmlv逆转录酶突变体及其应用
WO2023115314A1 (zh) 一种基于CRISPR-Cas系统和荧光共振能量转移的核酸检测方法
US20120135472A1 (en) Hot-start pcr based on the protein trans-splicing of nanoarchaeum equitans dna polymerase
CN111433373B (zh) Dna聚合酶
CN116096872A (zh) 热稳定的末端脱氧核苷酸转移酶
JP4249196B2 (ja) フラップエンドヌクレアーゼ
CN110577981A (zh) 一种基于人工模拟核酸的分子信标
CN114058735A (zh) 一种基于crispr技术检测手足口病的方法
US20230220449A1 (en) Marine dna polymerase i

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23859337

Country of ref document: EP

Kind code of ref document: A1