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CN-116751762-B - Cas12b proteins, single stranded guide RNAs, gene editing systems comprising same and related applications

CN116751762BCN 116751762 BCN116751762 BCN 116751762BCN-116751762-B

Abstract

The invention relates to the technical field of gene editing, in particular to a Cas12b mutant protein, a single-stranded guide RNA, a CRISPR/Cas12 gene editing system adopting the same and application thereof. More specifically, the complex formed by the Cas12b mutant protein and the single-stranded guide RNA can accurately locate a target DNA sequence and cut the target sequence, so that double-strand break damage occurs to the target sequence, has high specificity, can reduce the off-target rate of gene editing in cells or in vitro, can distinguish single base difference of target sites, and 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 Cas12b mutant protein comprising only a mutation at one amino acid residue R453A or D496A corresponding to the wild type ChCas b protein as set forth in SEQ ID No. 1.
  2. 2. A conjugate, the conjugate comprising: a) The Cas12b mutant protein of claim 1; b) Modifying moiety, and C) A linker for connecting the Cas12b 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 (NLS) sequence, cytosine deaminase (CBE), adenine deaminase (ABE), 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 Cas12b mutant protein of claim 1; b) Additional proteins and polypeptides, and C) A linker for linking the Cas12b 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 (NLS) sequence, cytosine deaminase (CBE), adenine deaminase (ABE), 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. A single-stranded guide RNA comprising a scaffold sequence and a CRISPR spacer sequence from the 5 'end to the 3' end, the scaffold sequence comprising a tracrRNA and a repeat sequence from the 5 'end to the 3' end, and the single-stranded guide RNA being a nucleic acid sequence obtained by truncation or mutation on the basis of SEQ ID No. 2; Wherein the truncated nucleic acid sequence is the nucleic acid sequence shown in SEQ ID NO. 4; wherein the mutation refers to the mutation of the 78 th nucleotide of the nucleic acid sequence shown in SEQ ID NO. 2 to generate A > G.
  7. 7. The single stranded guide RNA of claim 6, wherein the CRISPR spacer sequence is a sequence 19-28 nucleotides in length and capable of complementary pairing with a target sequence.
  8. 8. The single stranded guide RNA of claim 6, wherein the CRISPR spacer sequence is a sequence 24 nucleotides in length capable of complementary pairing to a target sequence.
  9. 9. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding: a) The Cas12b mutant protein of claim 1; b) The conjugate of claim 2 or 3, or C) The fusion protein of claim 4 or 5.
  10. 10. The isolated nucleic acid molecule of claim 9, further comprising a nucleic acid sequence encoding the single stranded guide RNA of any one of claims 6-8 or a nucleic acid sequence encoding a single stranded guide RNA comprising the scaffold sequence set forth in SEQ ID No. 2.
  11. 11. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding the single stranded guide RNA of any one of claims 6-8.
  12. 12. A vector comprising a nucleic acid sequence encoding: a) The Cas12b mutant protein of claim 1; b) The conjugate of claim 2 or 3, or C) The fusion protein of claim 4 or 5.
  13. 13. The vector of claim 12, wherein the vector is a plasmid vector.
  14. 14. The vector of claim 12, 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.
  15. 15. The vector according to any one of claims 12 to 14, further comprising a nucleic acid sequence encoding the single stranded guide RNA of any one of claims 6 to 8 or a nucleic acid sequence encoding a single stranded guide RNA comprising the scaffold sequence set forth in SEQ ID No. 2.
  16. 16. A vector comprising a nucleic acid sequence encoding the single stranded guide RNA of any one of claims 6-8.
  17. 17. A CRISPR/Cas12b gene editing system, comprising: 1) A protein component comprising: a) The Cas12b mutant protein of claim 1; b) The conjugate of claim 2 or 3, or C) The fusion protein of claim 4 or 5; d) A wild type ChCas b protein, conjugate or fusion protein as shown in SEQ ID NO. 1; 2) The single stranded guide RNA of any one of claims 6-8, or a single stranded guide RNA comprising the scaffold sequence shown in SEQ ID No. 2; wherein said protein component, when it is d) a wild-type ChCas b protein as set forth in SEQ ID NO. 1, a conjugate or fusion protein thereof, is capable of being used only in combination with the single stranded guide RNA of any one of claims 6-8; and, the protein component and the single stranded guide RNA bind to each other to form a complex.
  18. 18. A cell comprising the isolated nucleic acid molecule of any one of claims 9-11, or the vector of any one of claims 12-16.
  19. 19. The cell of claim 18, wherein the cell is a prokaryotic cell or a eukaryotic cell, the eukaryotic cell being an animal cell.
  20. 20. The cell of claim 19, wherein the animal cell is a mammalian cell.

Description

Cas12b proteins, single stranded guide RNAs, gene editing systems comprising same and related applications Technical Field The invention relates to the technical field of gene editing, in particular to a Cas12b mutant protein, a single-stranded guide RNA combined with the protein, a CRISPR/Cas12b gene editing system containing the same and related applications thereof. Background The CRISPR/Cas12b system is an adaptive immune system that bacteria and archaea evolve to protect against foreign virus or plasmid invasion. The CRISPR/Cas12b system contains a tracrRNA (trans-ACTIVATING RNA) and a crRNA (CRISPR-DERIVED RNA) that together form a complex with Cas12b to function. 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. Besides basic scientific research, the CRISPR/Cas12b gene editing system also has wide clinical application prospect. The most concern when using CRISPR/Cas gene editing systems for gene therapy is the problem of off-target. Off-target may disrupt normal genes, leading to cancer. Most CRISPR/Cas have off-target effects. Therefore, there is an urgent need to improve specificity and reduce off-target. In addition, many dominant mutations are caused by single base mutations. If the mutated allele can be knocked out, the therapeutic effect can be achieved by only retaining the normal allele. This requires that the CRISPR/Cas system be able to distinguish single base mutations. Most CRISPR/Cas systems currently cannot distinguish single base mutations. Disclosure of Invention The present inventors have found mutation sites related to cleavage specificity of wild-type ChCas b protein by repeated studies, thereby obtaining a series of Cas12b mutant proteins, all of which can constitute a CRISPR/Cas12b gene editing system with improved cleavage specificity that efficiently performs 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 Cas12b mutant protein comprising a mutation at one or more amino acid residues corresponding to R453, D496 and R1137 of a wild-type ChCas b protein. In a second aspect, the present invention provides a conjugate comprising: a) The Cas12b mutant protein of the first aspect; b) Modifying moiety, and C) Optionally a linker for linking the Cas12b mutant protein to the modifying moiety. In a third aspect, the present invention provides a fusion protein comprising: a) The Cas12b mutant protein of the first aspect; b) Additional proteins and polypeptides, and C) Optionally a linker for linking the Cas12b mutant protein to the additional proteins and polypeptides. In a fourth aspect, the invention provides a single stranded guide RNA comprising from 5 'to 3' a scaffold sequence comprising from 5 'to 3' a tracrRNA and a repeat sequence, and a CRISPR spacer sequence, and having a nucleic acid sequence that is truncated or mutated relative to the nucleic acid sequence set out in SEQ ID No. 2. In a fifth aspect, the invention provides an isolated nucleic acid molecule comprising a nucleic acid sequence encoding: a) The Cas12b 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 an isolated nucleic acid molecule comprising a nucleic acid sequence encoding the single stranded guide RNA of the fourth aspect. In a seventh aspect, the invention provides a vector comprising a nucleic acid sequence encoding: a) The Cas12b mutant protein of the first aspect; b) The conjugate according to the second aspect, or C) The fusion protein of the third aspect. In an eighth aspect, the invention provides a vector comprising a nucleic acid sequence encoding the single stranded guide RNA of the fourth aspect. In a ninth aspect, the present invention provides a CRISPR/Cas12b gene editing system comprising: 1) A protein component comprising: a) The Cas12b mutant protein of the first aspect; b) The conjugate according to the second aspect, or C) The fusion protein of the third aspect; d) Wild-type ChCas b protein, conjugate or fusion protein; 2) The nucleic acid sequence of the single stranded guide RNA of the fourth aspect, or a single stranded guide RNA comprising the scaffold sequence shown in SEQ ID NO. 5; Wherein said protein component can only be used in combination with the single stranded guide RNA of the fourth aspect when it is d) wild-type ChCas b protein, conjugate or fusion protein thereof; and,