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CN-116804190-B - SlugCas9 mutant proteins and related uses thereof

CN116804190BCN 116804190 BCN116804190 BCN 116804190BCN-116804190-B

Abstract

The invention relates to the technical field of gene editing, in particular to Slug mutant protein, a CRISPR/Cas9 gene editing system comprising the same and application thereof. More specifically, the compound formed by SlugCas mutant protein and single-stranded guide RNA is simpler than PAM of a wild SlugCas compound, has a larger targeting range, can accurately position a target DNA sequence and generate cutting, ensures that the target sequence is subjected to double-strand break damage, has high specificity, can reduce the off-target rate of gene editing in cells or in vitro, and has wide application prospect.

Inventors

  • WANG YONGMING
  • QI TAO

Assignees

  • 复旦大学

Dates

Publication Date
20260505
Application Date
20230612

Claims (20)

  1. 1. A SlugCas9 mutant protein comprising only a combination of mutations at a plurality of amino acid residues corresponding to the Q782, S888, L906, N984, E1012 and K1016 of the wild type SlugCas protein as set forth in SEQ ID No. 1: 1) Q782R, S888R, L906R, E1012K and K1016I, or 2) Q782R, S888R, L906R, N984S, E1012K and K1016I.
  2. 2. A conjugate, the conjugate comprising: a) The SlugCas mutant protein of claim 1; b) A modifying moiety; c) A linker for linking the SlugCas mutant protein to the modification; 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 SlugCas mutant protein of claim 1; b) Additional proteins and polypeptides; c) A linker for linking the SlugCas 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 SlugCas 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, further comprising a nucleic acid sequence encoding a single stranded guide RNA comprising a CRISPR spacer sequence and a scaffold sequence from the 5 'end to the 3' end.
  8. 8. The isolated nucleic acid molecule of claim 7, wherein the scaffold sequence has the nucleic acid sequence set forth in any one of SEQ ID NOs 2-4.
  9. 9. The isolated nucleic acid molecule of claim 7, wherein the CRISPR spacer sequence is a sequence 15-28 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 21 nucleotides in length and capable of complementary pairing with a target sequence.
  11. 11. A vector comprising a nucleic acid sequence encoding: a) The SlugCas 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, further comprising a nucleic acid sequence encoding a single stranded guide RNA comprising, from 5 'to 3' end, a CRISPR spacer sequence and a scaffold sequence.
  15. 15. The vector of claim 14, wherein the scaffold sequence has a nucleic acid sequence as set forth in any one of SEQ ID NOs 2-4.
  16. 16. The vector of claim 14, wherein the CRISPR spacer sequence is a sequence 15-28 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 21 nucleotides in length capable of complementary pairing with a target sequence.
  18. 18. A CRISPR/Cas9 gene editing system, comprising: 1) A protein component comprising: a) The SlugCas 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, from the 5 'end to the 3' end, a CRISPR spacer sequence and a scaffold sequence; and, the protein component and the single stranded guide RNA bind to each other to form a complex.
  19. 19. The CRISPR/Cas9 gene editing system according to claim 18, wherein the scaffold sequence has the nucleic acid sequence set forth in any one of SEQ ID NOs 2-4.
  20. 20. The CRISPR/Cas9 gene editing system according to claim 18, wherein the CRISPR spacer sequence is a sequence 15-28 nucleotides in length and capable of complementary pairing with a target sequence.

Description

SlugCas9 mutant proteins and related uses thereof Technical Field The invention relates to the technical field of gene editing, in particular to SlugCas mutant protein, a CRISPR/Cas9 gene editing system containing the same and related application for gene editing. Background The CRISPR/Cas9 system is an adaptive immune system that bacteria and archaea evolve to protect against foreign virus or plasmid invasion. The CRISPR/Cas9 system contains a tracrRNA (trans-ACTIVATING RNA) and a crRNA (CRISPR-DERIVED RNA) that function in concert with Cas9 to form a complex. the tracrRNA and crRNA can be fused into single guide RNA (sgRNA) by a linker sequence. 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/Cas9 system can target site sequences with a protospacer adjacent sequence (PAM) at the 3' end, the simpler the PAM requirement of the CRISPR/Cas9 system, the greater the targeting range of the CRISPR/Cas9 system. Besides basic scientific research, the CRISPR/Cas9 gene editing system also has wide clinical application prospect. Another key issue when using CRISPR/Cas9 gene editing systems for gene therapy is the off-target problem. Off-target may disrupt normal genes, leading to cancer. Most CRISPR/Cas9 have off-target effects. Therefore, there is a need to develop a CRISPR/Cas9 system with simpler PAM requirements and higher specificity to expand the scope of gene editing, reduce the occurrence of off-target, and increase the safety of gene editing. Disclosure of Invention The present inventors have found mutation sites related to PAM recognition, targeting specificity and cleavage activity of wild-type SlugCas protein through repeated studies, thereby obtaining a series of SlugCas mutant proteins, which can all constitute a CRISPR/Cas9 gene editing system with improved cleavage specificity that can efficiently perform gene editing with single-stranded guide RNAs, and thus completed the present invention. In summary, in a first aspect of the invention, there is provided a SlugCas, slugCas mutant protein comprising a mutation at one or more amino acid residues of Q782, S888, L906, N984, E1012 and K1016 corresponding to a wild type SlugCas protein. In a second aspect, the present invention provides a conjugate comprising: a) The SlugCas mutant protein of the first aspect; b) Modifying moiety, and C) Optionally a linker for linking the SlugCas mutant protein to the modification. In a third aspect, the present invention provides a fusion protein comprising: a) The SlugCas mutant protein of the first aspect; b) Additional proteins and polypeptides, and C) Optionally a linker for linking the SlugCas 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 SlugCas 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 SlugCas 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/Cas9 gene editing system comprising: 1) A protein component comprising: a) The SlugCas mutant protein of the first aspect; b) The conjugate according to the second aspect, or C) The fusion protein of the third aspect; And 2) A single stranded guide RNA, wherein the single stranded guide RNA comprises a CRISPR spacer sequence and a scaffold sequence from the 5 'end to the 3' end; and, the protein component and the single stranded guide RNA bind 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 SlugCas 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; (3) The carrier according to the fifth aspect, and (4) The CRISPR/Cas9 gene editing system of the sixth aspect; Wherein the single stranded guide RNA comprises a CRISPR spacer sequence and a sc