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CN-122012460-A - AnaCas9 mutant and gene editing system and application thereof

CN122012460ACN 122012460 ACN122012460 ACN 122012460ACN-122012460-A

Abstract

The invention relates to AnaCas mutant, a gene editing system and application thereof, and relates to the field of biotechnology. The AnaCas mutant is modified at a specific site, so that the gene editing activity of the AnaCas mutant is obviously improved, and the AnaCas mutant can be widely applied to high-efficiency gene editing of prokaryotes and eukaryotes, and especially can realize high-efficiency gene editing in mammalian cells (including human cells) and saccharomycetes.

Inventors

  • HUANG JUNJIU
  • ZHANG ZEMING

Assignees

  • 中山大学

Dates

Publication Date
20260512
Application Date
20260123

Claims (11)

  1. 1. A AnaCas mutant, characterized in that the AnaCas mutant comprises at least 1 site of amino acid differences ：E44、E95、N97、N101、E134、E137、D138、D139、D142、E143、E145、D186、D193、D194、D195、E196、D249、D252、A344、E345、E348、E349、H426、G428、N432、N459、E471、E482、D484、Q877、S878、D888、D895、E897、D898、E910、D914、K916、D928、K930、L936、K976、E979、E980、I992、K993、A994、V1006、D1009、L1012、D1015 or D1016 as compared to the wild-type Cas9 protein; The amino acid sequence of the wild Cas9 protein is shown as SEQ ID NO. 1.
  2. 2. The AnaCas mutant according to claim 1, wherein the AnaCas mutant has an amino acid difference ：E44K、E44Q、E95K、E95G、N97K、N97A、N101K、N101A、E134K、E134G、E137Q、D138K、D138N、D138G、D139K、D139G、D142K、D142N、E143K、E143Q、E143G、E145K、E145Q、E145G、D186N、D193K、D193G、D194G、D195K、D195N、E196K、E196G、D249K、D249G、D252K、D252N、D252G、A344K、E345Q、E348K、E349Q、E349G、H426K、G428K、N432K、N459K、E471Q、E482K、D484N、Q877K、S878K、D888K、D895K、D895N、D895G、E897K、E897Q、E897G、D898K、D898N、E910Q、D914N、K916R、D928K、K930R、L936N、K976R、E979K、E979Q、E980K、I992K、K993R、A994S、V1006K、D1009N、L1012K、D1015K、D1015N、D1015G、D1016N or D1016G of at least 1 site below.
  3. 3. The AnaCas mutant according to claim 2, wherein the AnaCas mutant has an amino acid difference of at least 1 position E44Q, E145G, D193K, D195K, D895K, D914N, D1016G, D895N, E897Q, D898N, D1015K, D N, or D1015G.
  4. 4. A guide RNA which, by binding to the AnaCas mutant of any one of claims 1 to 3, forms a CRISPR complex which is directed to bind specifically to a target sequence, said guide RNA comprising a guide sequence and a backbone sequence engineered from the sequence shown in SEQ ID No. 5, said engineering comprising truncation and/or mutation, said backbone sequence being as shown in any one of SEQ ID nos. 6 to 22.
  5. 5. The guide RNA of claim 4, wherein the backbone sequence is set forth in any one of SEQ ID NOs 6, 8, 9, 10, 12, 13, 18, 19, 21, 22.
  6. 6. The guide RNA of claim 5, wherein the engineering comprises: Modification (1) truncating the 1 st stem-loop structure from the 5 'end to the 3' end of the sequence shown in SEQ ID NO. 5; And/or modification (2) deleting the last stem-loop structure from the 5 'end to the 3' end of the sequence shown in SEQ ID NO. 5; the skeleton sequences are shown as SEQ ID NO. 8, 10, 21 and 22.
  7. 7. A CRISPR-Cas9 system, characterized in that it comprises a Cas9 protein and the guide RNA of any one of claims 4-6, which guides the CRISPR complex to specifically bind to a target sequence by binding to the Cas9 protein to form a CRISPR complex; the Cas9 protein comprises a fusion protein, a conjugate, a synthetic Cas9 protein comprising the amino acid sequence of the AnaCas mutant of any one of claims 1-3 or the AnaCas mutant of any one of claims 1-3, the fusion protein or conjugate comprising the AnaCas mutant linked to a homologous or heterologous functional domain.
  8. 8. An expressed gene encoding the AnaCas mutant of any one of claims 1-3, the guide RNA of any one of claims 4-6, or the Cas9 protein in the CRISPR-Cas9 system of claim 7.
  9. 9. An expression vector comprising the CRISPR-Cas9 system of claim 7, or the expressed gene of claim 8.
  10. 10. A recombinant cell comprising the AnaCas mutant of any one of claims 1-3, the guide RNA of any one of claims 4-6, the Cas9 protein in the CRISPR-Cas9 system of claim 7, the expression gene of claim 8, or the expression vector of claim 9.
  11. 11. Use of the AnaCas mutant of any one of claims 1-3, the guide RNA of any one of claims 4-6, the Cas9 protein in the CRISPR-Cas9 system of claim 7, the expression gene of claim 8, or the expression vector of claim 9 in a recombinant cell, pharmaceutical composition, or kit.

Description

AnaCas9 mutant and gene editing system and application thereof Technical Field The invention relates to AnaCas mutant, a gene editing system and application thereof, and relates to the field of biotechnology. Background Since the CRISPR/Cas9 system was developed as a gene editing tool, it shows excellent performance in scientific and clinical research and is the most widely used gene editing tool. The CRISPR/Cas9 serving as a gene editing tool can finish the targeting of a specific position of a DNA sequence only by using Cas9 protein and corresponding guide RNA (gRNA), and has the advantages of simplicity, high efficiency, good specificity and the like. The most widely studied application at present comprises Streptococcus pyogenes (SpCas 9) and Staphylococcus aureus (SaCas 9), but the larger volume of the SpCas9 and the more complex PAM sequence of the SaCas9 limit the application of the SpCas9, and the identification of the novel Cas9 protein with the smaller volume and the simple PAM sequence is beneficial to the development of gene editing work in scientific research and research. Disclosure of Invention Compared with wild AnaCas protein, the AnaCas mutant provided by the invention has obviously improved gene editing activity, can be widely applied to gene editing of prokaryotes and eukaryotes, and can realize efficient gene editing especially in mammalian cells (including human cells) and saccharomycetes. The invention provides a AnaCas mutant, the AnaCas mutant compared with wild type Cas9 protein, comprising the following amino acid differences ：E44、E95、N97、N101、E134、E137、D138、D139、D142、E143、E145、D186、D193、D194、D195、E196、D249、D252、A344、E345、E348、E349、H426、G428、N432、N459、E471、E482、D484、Q877、S878、D888、D895、E897、D898、E910、D914、K916、D928、K930、L936、K976、E979、E980、I992、K993、A994、V1006、D1009、L1012、D1015 or D1016 of at least 1 site; The amino acid sequence of the wild Cas9 protein is shown as SEQ ID NO. 1. In one embodiment, the amino acid sequence of the AnaCas mutant includes a sequence that has at least 70%, at least 80%, at least 85%, at least 90%, 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%, at least 99.9%, or 100% sequence identity compared to SEQ ID No. 1. In a specific embodiment of the invention, the amino acid difference is that the amino acid substitution at the site is any other amino acid, or that the amino acid at the site is absent. In particular embodiments of the invention, the AnaCas mutant has an amino acid sequence that has at least 70%, at least 80%, at least 85%, at least 90%, 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%, at least 99.9%, or 100% sequence identity compared to SEQ ID No. 1. In one embodiment, the AnaCas mutant has an amino acid difference ：E44K、E44Q、E95K、E95G、N97K、N97A、N101K、N101A、E134K、E134G、E137Q、D138K、D138N、D138G、D139K、D139G、D142K、D142N、E143K、E143Q、E143G、E145K、E145Q、E145G、D186N、D193K、D193G、D194G、D195K、D195N、E196K、E196G、D249K、D249G、D252K、D252N、D252G、A344K、E345Q、E348K、E349Q、E349G、H426K、G428K、N432K、N459K、E471Q、E482K、D484N、Q877K、S878K、D888K、D895K、D895N、D895G、E897K、E897Q、E897G、D898K、D898N、E910Q、D914N、K916R、D928K、K930R、L936N、K976R、E979K、E979Q、E980K、I992K、K993R、A994S、V1006K、D1009N、L1012K、D1015K、D1015N、D1015G、D1016N or D1016G of at least 1 site below. In one embodiment, the AnaCas9 mutant has an amino acid difference of at least 1 position E44Q, E145G, D193K, D195K, D895K, D914N, D1016G, D N, E897Q, D898N, D1015K, D1015N, or D1015G. In some embodiments of the invention, the AnaCas mutant can form a CRISPR complex with a guide RNA (gRNA). In some embodiments of the invention, the AnaCas mutant can form a CRISPR complex with a guide RNA (gRNA) that directs sequence-specific binding of the CRISPR complex to a target sequence. In some embodiments of the invention, the AnaCas mutant can form a CRISPR complex with a guide RNA (gRNA) that comprises a guide sequence engineered to direct sequence-specific binding of the CRISPR complex to a target sequence. In some embodiments of the invention, the AnaCas mutant can form a CRISPR complex with a guide RNA (gRNA) that directs the CRISPR complex to sequence specifically bind and cleave a target sequence. Optionally, the target sequence is double-stranded DNA, and optionally, the cleavage target sequence is only 1 single strand in the double-stranded DNA, or the cleavage target sequence is 2 single strands in the double-stranded DNA. In some embodiments of the invention, the AnaCas mutant can form a CRISPR complex with a guide RNA (gRNA) that directs the CRISPR complex sequence to specifically bind to a target sequence and cause double strand breaks in the target sequence to be cleaved. In a specific embodiment of the invention, the AnaCas