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CN-122012463-A - CRISPR enzyme, system and application

CN122012463ACN 122012463 ACN122012463 ACN 122012463ACN-122012463-A

Abstract

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

Inventors

  • LI SHANSHAN
  • ZHAO QINGZHI
  • REN YAJUN
  • LIU RUIHENG

Assignees

  • 山东舜丰生物科技有限公司

Dates

Publication Date
20260512
Application Date
20240828
Priority Date
20230829

Claims (10)

  1. 1. A Cas protein, characterized in that the Cas protein is any one of the following I-III: I. The amino acid sequence of the Cas protein 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%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity as compared to any of SEQ ID No.8, SEQ ID No.5, SEQ ID No.6, SEQ ID No.2, SEQ ID No.1, SEQ ID No.3, or SEQ ID No.4, and substantially retains the biological function of the sequence from which it is derived. II. The amino acid sequence of the Cas protein has a sequence of substitution, deletion or addition of one or more amino acids as compared with any one of SEQ ID No.8, SEQ ID No.5, SEQ ID No.6, SEQ ID No.2, SEQ ID No.1, SEQ ID No.3 or SEQ ID No.4, and substantially retains the biological function of the sequence from which it is derived; III, the Cas protein comprises any one of the amino acid sequences shown in SEQ ID No.8, SEQ ID No.5, SEQ ID No.6, SEQ ID No.2, SEQ ID No.1, SEQ ID No.3 or SEQ ID No. 4.
  2. 2. A fusion protein comprising the Cas protein of claim 1 and other modifying moieties.
  3. 3. An isolated polynucleotide, wherein the polynucleotide is a polynucleotide sequence encoding the Cas protein of claim 1 or a polynucleotide sequence encoding the fusion protein of claim 2.
  4. 4. A carrier, characterized in that, the vector comprising the polynucleotide of claim 3 operably linked to regulatory elements.
  5. 5. A CRISPR-Cas system, characterized in that the system comprises the Cas protein of claim 1 and at least one gRNA capable of binding to the Cas protein, the gRNA comprising a region that binds to the Cas protein and a targeting sequence of a targeting nucleic acid.
  6. 6. A composition, characterized in that it comprises: (i) A protein component selected from the Cas protein of claim 1 or the fusion protein of claim 2; (ii) A nucleic acid component selected from the group consisting of a gRNA, or a nucleic acid encoding the gRNA, or a precursor RNA of the gRNA, or a precursor RNA nucleic acid encoding the gRNA, the gRNA comprising a region that binds to the Cas protein of claim 1, and a targeting sequence of a targeting nucleic acid; The protein component and the nucleic acid component are bound to each other to form a complex.
  7. 7. An engineered host cell comprising the Cas protein of claim 1, or the fusion protein of claim 2, or the polynucleotide of claim 3, or the vector of claim 4, or the CRISPR-Cas system of claim 5, or the composition of claim 6.
  8. 8. Use of the Cas protein of claim 1, or the fusion protein of claim 2, or the polynucleotide of claim 3, or the vector of claim 4, or the CRISPR-Cas system of claim 5, or the composition of claim 6, or the host cell of claim 7 in gene editing, gene targeting, gene cleaving, cleaving double-stranded DNA, single-stranded DNA, or single-stranded RNA, non-specific cleavage and/or degradation of collateral nucleic acid, non-specific cleavage of single-stranded nucleic acid, nucleic acid detection, specific editing of double-stranded nucleic acid, base editing of single-stranded nucleic acid; Or in the preparation of a preparation or kit for gene editing, gene targeting, gene cleavage, cleavage of double-stranded DNA, single-stranded DNA or single-stranded RNA, nonspecific cleavage and/or degradation of collateral nucleic acid, nonspecific cleavage of single-stranded nucleic acid, nucleic acid detection, specific editing of double-stranded nucleic acid, base editing of single-stranded nucleic acid.
  9. 9. A method of editing, targeting, or cleaving a target nucleic acid, the method being a method of non-disease diagnosis and treatment, the method comprising contacting the target nucleic acid with the Cas protein of claim 1, or the fusion protein of claim 2, or the polynucleotide of claim 3, or the vector of claim 4, or the CRISPR-Cas system of claim 5, or the composition of claim 6, or the host cell of claim 7.
  10. 10. A method of detecting a target nucleic acid in a sample, the method being a method for non-disease diagnosis and treatment purposes, the method comprising contacting the sample with the Cas protein of claim 1, a gRNA (guide RNA) comprising a region that binds to the Cas protein and a guide sequence that hybridizes to the target nucleic acid, and a single stranded nucleic acid detector, detecting a detectable signal generated by cleavage of the single stranded nucleic acid detector by the Cas protein, thereby detecting the target nucleic acid, the single stranded nucleic acid detector not hybridizing to the gRNA.

Description

CRISPR enzyme, system and application The application is a divisional application of Chinese patent application with the application number 202411188361.3, the application date 2024, 8 months and 28 days, and the name of CRISPR enzyme and system and application. The present application claims priority from chinese patent application CN202311097808.1, whose filing date is 2023, 8, 29. The present application incorporates the entirety of the above-mentioned chinese patent application. Technical Field The invention relates to the field of gene editing, in particular to the technical field of regular clustered interval short palindromic repeat (CRISPR). Specifically, the invention screens out a novel class of Cas enzymes, and develops a corresponding gene editing tool and application thereof based on the novel Cas enzymes. Background CRISPR/Cas technology is a widely used gene editing technology that uses RNA-guided specific binding of target sequences on the genome and cleavage of DNA to create double strand breaks, site-directed gene editing using biological non-homologous end joining or homologous recombination. The CRISPR/Cas9 system is the most commonly used type II CRISPR system that recognizes the PAM motif of 3' -NGG and blunt-ends the target sequence. The CRISPR/Cas Type V system is a newly discovered class of CRISPR systems that have a 5' -TTN motif that performs cohesive end cleavage of a target sequence, e.g., cpf1, C2C1, casX, casY. However, the different CRISPR/Cas currently in existence each have different advantages and disadvantages. For example, cas9, C2C1 and CasX each require two RNAs for guide RNAs, whereas Cpf1 requires only one guide RNA and can be used for multiplex gene editing. CasX has a size of 980 amino acids, whereas common Cas9, C2C1, casY and Cpf1 are typically around 1300 amino acids in size. In addition, PAM sequences of Cas9, cpf1, casX, casY are all relatively complex and diverse, while C2C1 recognizes the stringent 5' -TTN, so its target site is easily predicted compared to other systems, thereby reducing potential off-target effects. In summary, given that the currently available CRISPR/Cas systems are limited by several drawbacks, the development of a more robust new CRISPR/Cas system with versatile good performance is of great importance for the development of biotechnology. Disclosure of Invention The inventors of the present application have unexpectedly discovered a novel endonuclease (Cas enzyme) through a number of experiments and repeated fumbling. Based on this finding, the present inventors developed a new CRISPR/Cas system and a gene editing method and a nucleic acid detection method based on the same. Cas effector proteins In one aspect, the invention provides a Cas protein, which is an effector protein in a CRISPR/Cas system, and in the invention, the Cas protein is called Cas-sf6102, cas-sf6103, cas-sf6117, cas-sf6119, cas-sf6126, cas-sf6127, cas-sf6129 and Cas-sf6132, and the amino acid sequences of the proteins are respectively shown in SEQ ID nos. 1 to 8. In one embodiment, the Cas protein amino acid sequence 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%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity compared to any of SEQ ID nos. 1-8, and substantially retains the biological function of the sequence from which it is derived. Preferably, the Cas protein is derived from the same species as Cas-sf6102, cas-sf6103, cas-sf6117, cas-sf6119, cas-sf6126, cas-sf6127, cas-sf6129, or Cas-sf 6132. In one embodiment, the Cas protein amino acid sequence has a sequence with one or more amino acid substitutions, deletions, or additions compared to any of SEQ ID nos. 1-8, and substantially retains the biological function of the sequence from which it is derived, including 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, or additions. Preferably, the Cas protein is derived from the same species as Cas-sf6102, cas-sf6103, cas-sf6117, cas-sf6119, cas-sf6126, cas-sf6127, cas-sf6129, or Cas-sf 6132. In some embodiments, the Cas protein of the present invention is capable of recognizing a pre-spacer adjacent motif (PAM), and the target nucleic acid comprises or consists of PAM. It will be apparent to those skilled in the art that the structure of a protein may be altered without adversely affecting its activity and functionality, for example, one or more conservative amino acid substitutions may be introduced into the amino acid sequence of the protein without adversely affecting the activity and/or three-dimensional structure of the protein molecule. Examples and embodiments of conservative amino acid substitutions are apparent to those skilled in the art. In particular, the amino acid residue may be substituted with a