Fusion protein that improves gene editing efficiency and application thereof

1 . A fusion protein for improving gene editing efficiency, comprising functional domain of a single-stranded DNA binding protein, a nucleoside deaminase and a nuclease,

wherein the nucleoside deaminase is located at the N terminal of the nuclease, and

wherein the functional domain of the single-stranded DNA binding protein is located between the nucleoside deaminase and the nuclease.

2 . The fusion protein according to claim 1 , wherein the non-sequence-specific single-stranded DNA binding protein is selected from any one of RPA70, RPA32, BRCA2, hnRNPK, PUF60 and Rad51; and

the sequence-specific single-stranded DNA binding protein is selected from any one of TEBP, Teb1 and POT1.

3 . The fusion protein according to claim 1 , wherein the functional domain of the single-stranded DNA binding protein comprises any one of the following four domains: OB fold, KH domain, RRMS and whirly domain of the single-stranded DNA binding protein.

4 . The fusion protein according to claim 1 , wherein the functional domain of the single-stranded DNA binding protein comprises DNA binding domain of Rad51 or DNA binding domain of RPA70.

5 . The fusion protein according to claim 4 , wherein amino acid sequence of the DNA binding domain of Rad51 comprises a sequence of SEQ ID NO: 1;

or, coding sequence of the DNA binding domain of Rad51 comprises a sequence of SEQ ID NO: 2.

6 . The fusion protein according to claim 4 , wherein amino acid sequence of the DNA binding domain of RPA70 comprises a sequence of SEQ ID NO: 11;

or, coding sequence of the DNA binding domain of RPA70 comprises a sequence of SEQ ID NO: 12.

7 . The fusion protein according to claim 1 , wherein the nucleoside deaminase comprises cytosine deaminase or adenosine deaminase.

8 . The fusion protein according to claim 7 , wherein the cytosine deaminase comprises rat-derived cytosine deaminase.

9 . The fusion protein according to claim 8 , wherein amino acid sequence of the rat-derived cytosine deaminase comprises a sequence of SEQ ID NO: 3;

or, coding sequence of the rat-derived cytosine deaminase comprises a sequence of SEQ ID NO: 4.

10 . The fusion protein according to claim 7 , wherein the cytosine deaminase comprises human-derived cytosine deaminase APOBEC3A.

11 . The fusion protein according to claim 10 , wherein the amino acid sequence of the human-derived cytosine deaminase APOBEC3A comprises a sequence of SEQ ID NO: 13;

or, coding sequence of the human-derived cytosine deaminase APOBEC3A comprises a sequence of SEQ ID NO: 14.

12 . The fusion protein according to claim 7 , wherein the cytosine deaminase comprises the mutant of the cytosine deaminase APOBEC3A, and the mutant mutates asparagine at position 57 of the cytosine deaminase APOBEC3A into glycine.

13 . The fusion protein according to claim 12 , wherein the cytosine deaminase APOBEC3A is derived from a human.

14 . The fusion protein according to claim 13 , wherein amino acid sequence of the cytosine deaminase APOBEC3A comprises the sequence of SEQ ID NO: 13;

or, coding sequence of the cytosine deaminase APOBEC3A comprises the sequence of SEQ ID NO: 14.

15 . The fusion protein according to claim 12 , wherein amino acid sequence of the mutant of the cytosine deaminase APOBEC3A comprises a sequence of SEQ ID NO: 15;

or, coding sequence of the cytosine deaminase APOBEC3A comprises a sequence of SEQ ID NO: 16.

16 . The fusion protein according to claim 1 , wherein the nuclease is selected from one or more of Cas9, Cas3, Cas8a, Cas8b, Cas10d, Cse1, Csy1, Csn2, Cas4, Cas10, Csm2, Cmr5, Fok1 and Cpf1.

17 . The fusion protein according to claim 16 , wherein the nuclease is Cas9.

18 . The fusion protein according to claim 17 , wherein the Cas9 is selected from Cas9 derived from Streptococcus pneumoniae, Staphylococcus aureus, Streptococcus pyogenes or Streptococcus thermophilus.

19 . The fusion protein according to claim 17 , wherein the Cas9 is selected from Cas9 mutants VQR-spCas9, VRER-spCas9 or spCas9n.

20 . The fusion protein according to claim 19 , wherein amino acid sequence of spCas9n comprises a sequence of SEQ ID NO: 5;

or, coding sequence of the spCas9n comprises a sequence of SEQ ID NO: 6.

21 . The fusion protein according to claim 1 , wherein the fusion protein further comprises an NLS.

22 . The fusion protein according to claim 21 , wherein the NLS is located at at least one terminal of the fusion protein.

23 . The fusion protein according to claim 21 , wherein amino acid sequence of the NLS comprises a sequence of SEQ ID NO: 7;

or, coding sequence of the NLS comprises a sequence of SEQ ID NO: 8.

24 . The fusion protein according to claim 1 , wherein the fusion protein further comprises more than two copies of UGI.

25 . The fusion protein according to claim 24 , wherein the UGI is located at at least one terminal of the fusion protein.

26 . The fusion protein according to claim 24 , wherein amino acid sequence of the UGI comprises a sequence of SEQ ID NO: 9;

or, coding sequence of the UGI comprises a sequence of SEQ ID NO: 10.

27 . A recombinant cell or recombinant bacterium containing the fusion protein according to claim 1 .

28 . The biomaterial according to claim 27 , wherein the cells are T cells, hematopoietic stem cells, bone marrow cells, red blood cells or red blood cell precursor cells.

29 . An sgRNA for gene editing of a target gene in cells, wherein the target sequence of the sgRNA comprises at least one of SEQ ID NO: 17-36.

30 . The sgRNA according to claim 29 , wherein the cells are T cells, hematopoietic stem cells, bone marrow cells, red blood cells or red blood cell precursor cells.

31 . The sgRNA according to claim 29 , wherein the target sequence is the promoter of HBG1 or HBG2.

32 . A single-base gene editing system, wherein the system comprises the fusion protein according to claim 1 , and a sgRNA, wherein the sgRNA guides the fusion protein to conduct single-base gene editing on a target gene in a target cell.

33 . The single-base gene editing system according to claim 32 , wherein the target sequence of the sgRNA comprises at least one of SEQ ID NO: 17-36;

or, the cells are T cells, hematopoietic stem cells, bone marrow cells, red blood cells or a red blood cell precursor cells,

or, the target sequence is at the promotor the promoter of HBG1 or HBG2.

34 . A method for preparing a product for gene editing, treating or preventing disease, animal model or new plant variety, which comprises using the fusion protein according to claim 1 to conduct gene editing in a subject, an animal or a plant,

wherein the disease is beta-hemoglobinopathy or Duchenne muscular dystrophy (DMD).

35 . The method according to claim 34 , wherein the beta-hemoglobinopathy comprises beta-thalassemia or sickle cell anemia.

36 . A method for single-base gene editing, which comprises the steps of introducing the fusion protein according to claim 1 and the sgRNA into cells to edit target gene, wherein the sgRNA guides the fusion protein to conduct single-base gene editing on target gene in target cell.

37 . The method according to claim 36 , wherein the target sequence of the sgRNA comprises at least one of SEQ ID NO: 17-36;

or, the cells are T cells, hematopoietic stem cells, bone marrow cells, red blood cells or red blood cell precursor cells;

or, the target sequence is the promoter of HBG1 or HBG2.

38 . A method for constructing animal models of disease, which comprises the steps of introducing the fusion protein according to claim 1 and the sgRNA into animal cells to conduct gene editing of the target gene, and

creating animal cells bearing the disease or an animal bearing the disease.

39 . The method according to claim 38 , wherein the target sequence of the sgRNA comprises SEQ ID NO: 35, and the target gene comprises an HBG gene.

40 . The method according to claim 38 , wherein the target sequence of the sgRNA comprises the sequence of SEQ ID NO: 36, and the target gene comprises a DMD gene.

41 . The method according to claim 38 , wherein the animals are mammals,

or, the cells are embryonic cells,

or, the method of introduction is one or any combination of vector transformation, microinjection, transfection, lipid transfection, heat shock, electroporation, transduction, gene gun, and DEAE-dextran mediated transfer,

or, the introduction is carried out using mRNA of the fusion protein according to claim 1 and the sgRNA.

42 . The method according to claim 41 , wherein the animals are rats or mice;

or, when the method of introduction is microinjection, the concentration of the mRNA of the fusion protein according to claim 1 for introduction is 1-1,000 ng/μL.

43 . The method according to claim 42 , wherein the concentration ratio of the mRNA of the fusion protein according to claim 1 to the sgRNA used for introduction is 1:(5-1).

44 . A method for treating beta-hemoglobinopathy or Duchenne muscular dystrophy (DMD), comprising:

administering to a subject in need thereof a delivery vector of the gene encoding the fusion protein according to claim 1 and a sgRNA.

45 . A product for treating beta-hemoglobinopathy or Duchenne muscular dystrophy (DMD), which comprises: delivery vector of the gene encoding the fusion protein according to claim 1 and a sgRNA,

wherein the sgRNA guides the fusion protein to conduct single-base gene editing on the target gene in the target cell; and the target sequence is the promoter of HBG1 or HBG2, or the target gene is DMD gene.

46 . The product according to claim 45 , wherein

the beta-hemoglobinopathy comprises beta thalassemia or sickle cell anemia;

or the cells are T cells, hematopoietic stem cells, bone marrow cells, red blood cells or red blood cell precursor cells.

47 . The product according to claim 45 , wherein the delivery vector comprises a viral vector or a non-viral vector;

wherein the viral vector comprises an adeno-associated viral vector, an adenoviral vector, a lentiviral vector, a retroviral vector or an oncolytic virus vector; and the non-viral vector comprises a cationic high-molecular polymer or a liposome.

48 . A fusion protein for improving gene editing efficiency, comprising functional domain of a single-stranded DNA binding protein, a nucleoside deaminase and a nuclease;

wherein the nucleoside deaminase is located at the N terminal of the nuclease;

wherein the functional domain of the single-stranded DNA binding protein is located between the nucleoside deaminase and the nuclease; and

wherein the single-stranded DNA binding protein is Rad51.

49 . A fusion protein for improving gene editing efficiency, comprising functional domain of a single-stranded DNA binding protein, a nucleoside deaminase and a nuclease;

wherein the nucleoside deaminase is located at the N terminal of the nuclease;

wherein the functional domain of the single-stranded DNA binding protein is located between the nucleoside deaminase and the nuclease; and

wherein the single-stranded DNA binding protein is RPA70.