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CN-117025570-B - Cas12a mutant protein, gene editing system containing Cas12a mutant protein and application

CN117025570BCN 117025570 BCN117025570 BCN 117025570BCN-117025570-B

Abstract

The invention relates to the technical field of gene editing, in particular to an Mb4Cas12a mutant protein, a CRISPR/Cas12a gene editing system adopting the mutant protein and application thereof. The complex formed by the Mb4Cas12a mutant protein and crRNA can accurately locate a target DNA sequence and cut the target sequence, so that double-strand break damage occurs to the target sequence, the specificity is high, the off-target rate of gene editing in cells or in vitro can be reduced, single base difference of target sites can be distinguished, and the complex has wide application prospect.

Inventors

  • WANG YONGMING
  • WEI JINGJING

Assignees

  • 复旦大学

Dates

Publication Date
20260505
Application Date
20230424

Claims (20)

  1. 1. A Cas12a mutant protein comprising only a mutation at one amino acid residue G257, N276, K291, and F370A corresponding to the wild-type Mb4Cas12a protein as set forth in SEQ ID NO:1, G257A, N276A, K291A or F370A.
  2. 2. A conjugate, the conjugate comprising: a) The Cas12a mutant protein of claim 1; b) A modifying moiety; c) A linker for connecting the Cas12a mutant protein and the modifying moiety; wherein the modifying moiety is selected from the group consisting of an additional protein or polypeptide, a detectable label, or a combination thereof; Wherein the additional protein or polypeptide is selected from one or more of an epitope tag, a reporter protein or nuclear localization signal sequence, cytosine deaminase, adenine deaminase, cytosine methylase DNMT3A and MQ1, cytosine demethylase Tet1, transcriptional activator proteins VP64, p65 and RTA, transcriptional repressor protein KRAB, histone acetylase p300, histone deacetylase LSD1, and endonuclease FokI.
  3. 3. The conjugate of claim 2, wherein the linker is a linker of 1-50 amino acids in length.
  4. 4. A fusion protein, the fusion protein comprising: a) The Cas12a mutant protein of claim 1; b) Additional proteins and polypeptides; c) A linker for linking the Cas12a mutant protein to the additional proteins and polypeptides; Wherein the additional protein or polypeptide is selected from one or more of an epitope tag, a reporter protein or nuclear localization signal sequence, cytosine deaminase, adenine deaminase, cytosine methylase DNMT3A and MQ1, cytosine demethylase Tet1, transcriptional activator proteins VP64, p65 and RTA, transcriptional repressor protein KRAB, histone acetylase p300, histone deacetylase LSD1, and endonuclease FokI.
  5. 5. The fusion protein of claim 4, wherein the linker is a 1-50 amino acid linker in length.
  6. 6. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding: a) The Cas12a mutant protein of claim 1; b) The conjugate of claim 2 or 3, or C) The fusion protein of claim 4 or 5.
  7. 7. The isolated nucleic acid molecule of claim 6, wherein the isolated nucleic acid molecule further comprises a nucleic acid sequence encoding a single-stranded guide RNA capable of being used in conjunction with the Cas12a mutant protein, the single-stranded guide RNA comprising a scaffold sequence and a CRISPR spacer sequence.
  8. 8. The isolated nucleic acid molecule of claim 7, wherein the scaffold sequence is the scaffold sequence shown in SEQ ID No. 2.
  9. 9. The isolated nucleic acid molecule of claim 7, wherein the CRISPR spacer sequence is a sequence 18-28 nucleotides in length and capable of complementary pairing with a target sequence.
  10. 10. The isolated nucleic acid molecule of claim 7, wherein the CRISPR spacer sequence is a sequence 24 nucleotides in length and capable of complementary pairing with a target sequence.
  11. 11. A vector comprising a nucleic acid sequence encoding: a) The Cas12a mutant protein of claim 1; b) The conjugate of claim 2 or 3, or C) The fusion protein of claim 4 or 5.
  12. 12. The vector of claim 11, wherein the vector is a plasmid vector.
  13. 13. The vector of claim 11, wherein the vector is a pUC19 vector, an adherent vector, a pAAV2_itr vector, a retroviral vector, a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector.
  14. 14. The vector of any one of claims 11 to 13, wherein the vector further comprises a nucleic acid sequence encoding a single stranded guide RNA capable of being used with the Cas12a mutant protein, the single stranded guide RNA comprising a scaffold sequence and a CRISPR spacer sequence.
  15. 15. The vector of claim 14, wherein the scaffold sequence is the scaffold sequence shown in SEQ ID No. 2.
  16. 16. The vector of claim 14, wherein the CRISPR spacer sequence is a sequence 18-28 nucleotides in length and capable of complementary pairing with a target sequence.
  17. 17. The vector of claim 14, wherein the CRISPR spacer sequence is a sequence 24 nucleotides in length that is capable of complementary pairing with a target sequence.
  18. 18. A CRISPR/Cas12a gene editing system, comprising: 1) A protein component comprising: a) The Cas12a mutant protein of claim 1; b) The conjugate of claim 2 or 3, or C) The fusion protein of claim 4 or 5; And 2) A single stranded guide RNA comprising a scaffold sequence and a CRISPR spacer sequence; and, the protein component and the single stranded guide RNA bind to each other to form a complex.
  19. 19. The CRISPR/Cas12a gene editing system according to claim 18, wherein the scaffold sequence is the scaffold sequence shown in SEQ ID No. 2.
  20. 20. The CRISPR/Cas12a gene editing system according to claim 18, wherein the CRISPR spacer sequence is a sequence 18-28 nucleotides in length that is capable of complementary pairing with a target sequence.

Description

Cas12a mutant protein, gene editing system containing Cas12a mutant protein and application Technical Field The invention relates to the technical field of gene editing, in particular to a Cas12a mutant protein, a CRISPR/Cas12a gene editing system containing the mutant protein and related applications thereof. Background The CRISPR/Cas system is an adaptive immune system that bacteria and archaea evolve to resist foreign virus or plasmid invasion, where CRISPR/Cas9 and CRISPR/Cas12 systems were developed as gene editing tools. These two systems can be further divided into several sub-classes. The CRISPR/Cas12a system belongs to a subclass of CRISPR/Cas12 and functions by virtue of crRNA (CRISPR-DERIVED RNA) forming a complex together with Cas12, without the inclusion of a tracrRNA (trans-ACTIVATING RNA). When DNA breaks and damage occurs, two major DNA damage repair mechanisms within the cell are responsible for repair, non-homologous end joining (NHEJ) and homologous recombination (homologous recombination, HR). The NHEJ repair results in base deletion or insertion, gene knockout can be performed, and the HR repair can be used for site-directed gene insertion and precise base substitution under the condition of providing a homologous template. The CRISPR/Cas12a has wide application prospect in basic scientific research and gene therapy. CRISPR/Cas12a is more specific than CRISPR/Cas9 and is not easily off-target. There is room for improvement in CRISPR/Cas12a specificity. CRISPR/Cas12a can tolerate single base mismatches between crRNA and target sequence. When there is a one base difference between two alleles in the genome, only one of the alleles cannot be knocked out. Many dominant mutations are caused by single base mutations, which require that the CRISPR/Cas12a system be able to distinguish single base mutations if the mutant allele can be specifically knocked out, which is not possible with most CRISPR/Cas12a systems today. Disclosure of Invention The present inventors have found mutation sites related to cleavage specificity of wild-type Mb4Cas12a protein by repeated studies, thereby obtaining a series of Cas12a mutant proteins, all of which can constitute a CRISPR/Cas12a gene editing system with improved cleavage specificity that efficiently performs gene editing with single-stranded guide RNAs, and completed the present invention. In summary, in a first aspect of the invention, there is provided a Cas12a mutant protein comprising a mutation at one or more amino acid residues corresponding to G257, N276, K291, and F370 of a wild-type Mb4Cas12a protein. In a second aspect, the present invention provides a conjugate comprising: a) The Cas12a mutant protein of the first aspect; b) Modifying moiety, and C) Optionally a linker for linking the Cas12a mutant protein to the modifying moiety. In a third aspect, the present invention provides a fusion protein comprising: a) The Cas12a mutant protein of the first aspect; b) Additional proteins and polypeptides, and C) Optionally a linker for linking the Cas12a mutant protein to the additional proteins and polypeptides. In a fourth aspect, the invention provides an isolated nucleic acid molecule comprising a nucleic acid sequence encoding: a) The Cas12a mutant protein of the first aspect; b) The conjugate according to the second aspect, or C) The fusion protein of the third aspect. In a fifth aspect, the invention provides a vector comprising a nucleic acid sequence encoding: a) The Cas12a mutant protein of the first aspect; b) The conjugate according to the second aspect, or C) The fusion protein of the third aspect. In a sixth aspect, the invention provides a CRISPR/Cas12a gene editing system comprising: 1) A protein component comprising: a) The Cas12a mutant protein of the first aspect; b) The conjugate according to the second aspect, or C) The fusion protein of the third aspect; 2) A single stranded guide RNA comprising a scaffold sequence and a CRISPR spacer sequence; And, the protein component and the nucleic acid component are bound to each other to form a complex. In a seventh aspect, the invention provides a cell comprising the isolated nucleic acid molecule of the fourth aspect, or the vector of the fifth aspect. In an eighth aspect, the present invention provides a method of gene editing of a target sequence in an intracellular or in vitro environment, the method comprising contacting any one of the following (1) to (4) with the target sequence in an intracellular or in vitro environment: (1) The Cas12a mutant protein of the first aspect, the conjugate of the second aspect, or the fusion protein of the third aspect, and a single stranded guide RNA; (2) The isolated nucleic acid molecule of the fourth aspect and optionally an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a single stranded guide RNA capable of being used in combination with the Cas12a mutant protein of the first aspect; (3) The vector of the fifth aspect