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CN-121065143-B - Base editor and application thereof

CN121065143BCN 121065143 BCN121065143 BCN 121065143BCN-121065143-B

Abstract

The invention discloses a base editor and application thereof, and provides a nucleic acid base editor, in particular to a base editor not based on CRISPR technology. The base editor includes a sequence-specific DNA binding protein, a nicking enzyme, an exonuclease, and a base-specific deaminase. The base editor has single-chain specificity, and compared with the traditional base editor, the base editor has wide applicability in cells, and can play a role in cell nucleus, mitochondrial DNA and/or chloroplast DNA. The base editor realizes high-efficiency base editing, has the characteristics of high purity of base editing products and few byproducts with indels, and is favorable for being used as a high-efficiency and safe gene editing tool.

Inventors

  • GAO CAIXIA
  • K.T.Zhao
  • SUN YU
  • HU JIACHENG
  • LI BOSHU

Assignees

  • 中国科学院遗传与发育生物学研究所
  • 北京齐禾生物科技有限公司

Dates

Publication Date
20260505
Application Date
20231130
Priority Date
20221215

Claims (20)

  1. 1. A nucleobase editor for use with an organelle, said nucleobase editor comprising the following components: a) A sequence-specific DNA binding protein; b) A nicking enzyme; c) An exonuclease; d) Base-specific deaminase, and E) A organelle targeting signal sequence; the sequence-specific DNA binding protein is a TALE protein; The incision enzyme is a dimer of FokI cutting functional domain monomers or a mutant thereof, the dimer or the mutant thereof consists of a pair of interactive FokI cutting functional domain monomers, and only one FokI cutting functional domain monomer in the dimer or the mutant thereof has DNA inscribing activity; The FokI cleavage functional domain monomer with DNA endo-activity is selected from FokI-L protein with an amino acid sequence shown as SEQ ID No.87 and FokI-R protein with an amino acid sequence shown as SEQ ID No. 88; The FokI cleavage functional domain monomer with the lost DNA endo-activity is selected from FokI-L D450A protein with an amino acid sequence shown as SEQ ID No. 60, fokI-L D467A protein with an amino acid sequence shown as SEQ ID No. 61, fokI-R D450A protein with an amino acid sequence shown as SEQ ID No. 62 or FokI-R D467A protein with an amino acid sequence shown as SEQ ID No. 63; the base-specific deaminase is selected from cytosine-specific deaminase or adenine-specific deaminase; the exonuclease is selected from 5 'exonuclease or 3' exonuclease; the organelle targeting signal sequence is selected from a mitochondrial targeting sequence MTS or a chloroplast transit peptide CTP.
  2. 2. The nucleobase editor of claim 1 wherein the components of the nucleobase editor are present alone or comprise one or more fusion proteins.
  3. 3. The nucleobase editor according to claim 1 or 2, wherein the amino acid sequence of the base specific deaminase is selected from the group consisting of SEQ ID nos. 36-59, 80-86.
  4. 4. The nucleobase editor of claim 1 or 2 wherein the base specific deaminase is a cytosine specific deaminase.
  5. 5. The nucleobase editor of claim 4 wherein the cytosine-specific deaminase is one or more of hAPOBEC A, rAPOBEC1, hAID, pmCDA1, or Sdd deaminase.
  6. 6. The nucleobase editor of claim 4, wherein the nucleobase editor further comprises: f) Uracil Glycosylase Inhibitors (UGIs); And the uracil glycosylase inhibitor is present alone or in combination with other nucleobase editor components to form at least one fusion protein.
  7. 7. The nucleobase editor of claim 1 or 2 wherein the base-specific deaminase is an adenine-specific deaminase.
  8. 8. The nucleobase editor of claim 7 wherein the adenine-specific deaminase is TadA-8e.
  9. 9. The nucleobase editor of any one of claims 1,2, 5-6, 8 further comprising: g) γb; The γb forms at least one fusion protein with other nucleobase editor components.
  10. 10. The nucleobase editor according to claim 1 or 2, characterized in that the amino acid sequence of the exonuclease is selected from the group consisting of SEQ ID nos. 64-67, 153.
  11. 11. A fusion protein as a nucleobase editor, characterized in that it comprises a protein domain of the nucleobase editor as defined in any one of claims 1-10.
  12. 12. A recombinant expression construct for nucleobase editing, wherein said recombinant expression construct is used for expressing the nucleobase editor of any one of claims 1-10 or the fusion protein of claim 11.
  13. 13. A method for nucleobase editing in a cell for non-therapeutic purposes, characterized in that the nucleobase editor according to any one of claims 1-10 or the recombinant expression construct according to claim 12 is introduced into a cell for editing a gene of interest.
  14. 14. The method of nucleobase editing according to claim 13, wherein said gene of interest is selected from mitochondrial genomic DNA or chloroplast genomic DNA.
  15. 15. Use of the base editor of any one of claims 1-10, the fusion protein of claim 11 or the recombinant expression construct of claim 12 for the preparation of a reagent for base editing of DNA in a cell, said cell being a mammalian cell, a bacterium, a protozoan, a fungus, an insect cell or a plant cell.
  16. 16. The use according to claim 15, wherein the plant cell is derived from the whole plant of a monocotyledonous or dicotyledonous plant.
  17. 17. The use according to claim 15, wherein the plant cells are derived from seedlings of monocotyledonous or dicotyledonous plants.
  18. 18. The use according to claim 15, wherein the plant cells are derived from a meristem, a basal tissue, a vascular tissue or a epithelial tissue of a monocotyledonous or dicotyledonous plant.
  19. 19. The use according to claim 15, wherein the plant cell is derived from a seed, leaf, root, bud, stem, flower or fruit of a monocot or dicot.
  20. 20. Use according to claim 15, wherein the plant cells are derived from stolons, bulbs, tubers, corms, shoots or asexual peripheral branches of monocotyledonous or dicotyledonous plants.

Description

Base editor and application thereof The application relates to a Chinese patent application with the application date of 2023, 11, 30, the application number of 202311622479.8 and the name of a base editor and application. PRIORITY AND RELATED APPLICATION The present application claims priority from chinese patent application 202211613160.4 entitled "a base editor and its use" filed on 12/15/2022 and chinese patent application 202311017698.3 entitled "a base editor and its use" filed on 8/14/2023, the entire contents of which including the appendix are incorporated herein by reference. Technical Field The invention relates to the field of gene editing, in particular to a nucleic acid base editor, and particularly relates to a base editor comprising sequence-specific DNA binding protein, nicking enzyme, exonuclease and base-specific deaminase and application thereof. Background Mutations in genomic and mitochondrial DNA are known to lead to various genetic diseases (Newby et al 2021, nature 595:295-302), and correction of these mutations is expected to be effective in treating or ameliorating some serious diseases. In plants, some important agronomic traits are related to Single Nucleotide Variations (SNVs) occurring in the plant genome, plant mitochondrial genome or plant chloroplast genome, and the introduction of these SNVs into plants can promote plant performance, molecular breeding, restore gene function to alleviate disease states, etc. Genome editing has shown great potential for genome modification, and targeted base substitution can be achieved without introducing DNA Double Strand Breaks (DSBs) in the genome editing tool by base editing, thereby achieving more accurate and precise editing (Gaudelli et al, 2017, nature 551:464-471; komor et al, 2016, nature 533:420-424), and thus having broad prospects in disease treatment and crop improvement. Cytosine Base Editors (CBE) (Komor et al., 2016, nature 533:420-424) and Adenine Base Editors (ABE) (Gaudelli et al., 2017, nature 551:464-471) are the most widely used base editors. In CBE systems, CRISPR-Cas9 nickase (nCas 9) with single-stranded DNA cleavage activity is directed by the sgRNA to the target dsDNA, nCas cleaves the sgRNA targeting strand, forming an R loop. Subsequently, single-strand specific cytosine deaminase converts cytosine (C) to uracil (U) within a window of about five nucleotides in the single-strand DNA bubble produced by nCas, which is replaced by T after DNA repair, resulting in a conversion of C: G base pairs to T: a base pairs. In addition, the addition of Uracil Glycosylase Inhibitors (UGIs) that have the effect of blocking uracil excision and its downstream processes can improve base editing efficiency and product purity. Cytosine deaminase enzymes suitable for Cas-mediated CBE systems include, but are not limited to apodec 1, hAID and hAPOBEC a. Recently, new deaminase systems have been found to be suitable for use in the deaminase of the present invention （Huang, J. et al. Discovery of new deaminase functions by structure-based protein clustering. bioRxiv (2023). ）. NCas9 was fused to a single-stranded DNA adenine deaminase TadA obtained by artificial evolution, yielding the ABE system (Gaudelli et al., 2017, nature 551:464-471). ABE works on a similar principle to CBE, nCas cleaves under the guidance of sgRNA to create a gap in the target strand of DNA, whereas adenine deaminase TadA converts adenine (A) to hypoxanthine (I), which is replaced by G after DNA repair, resulting in the conversion of A: T base pairs to G: C base pairs. However, the ABE system does not require UGI to increase its editing efficiency or product concentration because uracil intermediates are not involved in the process. The above mentioned ABEs and CBEs can work effectively in the nucleus but they cannot work in the chloroplasts or mitochondria because the sgrnas in the CRISPR system cannot transfer efficiently into these organelles. 2020. Over the years, researchers have developed a non-CRISPR base editor system consisting of protein components only. This new base editor system is designated DdCBE (Mok et al 2020, nature 583:631-637). DdCBE core components include double-stranded DNA cytosine deaminase DddA, which can convert C on double-stranded DNA to U without the need for CRISPR-Cas9 to create single-stranded DNA. However, the intact DddA is cytotoxic, so it is split in two halves-DddA-N and DddA-C, fused to a pair of TALE proteins, respectively. DddA-N and DddA-C recombine to restore their cytosine deaminase activity when directed to the target DNA sequence by TALE pairs, which, like the CRISPR-based CBE system, can also convert C: G base pairs to T: A base pairs, adding UGI can increase the base editing efficiency and product purity of DdCBE. Due to the whole protein component characteristic of DdCBE system, it can not only act in nucleus, but also be transported into chloroplast and mitochondria, realizing the targeted cytosine base editing