System and Method of Induced Mutant Protein Based on Activation-induced Cytidine Deaminase

Abstract

The present invention provides a mutant protein of activation-induced cytidine deaminase, wherein hsAID is mutated with the following mutations: T82I, K10E, K34E, E156G, 181*, S38, H130, V152, R174, T100. The present invention also provides a High-efficiency Base Editor, including the mutant protein of activation-induced cytidine deaminase of the present invention and a DNA-specific binding protein, which are linked sequentially via a linking sequence. The present invention also provides a single-base locus-directed editing system, including single-base locus-directed editing proteins and target Hyper Mutation Fragment. Compared with the existing activation-induced cytidine deaminase (AID)-based single-base editing system, the mutant protein inducing system of the present invention has a smaller molecular weight and higher mutation efficiency.

What is claimed is: 
     
         1 . A mutant protein of activation-induced cytidine deaminase, wherein hsAID is mutated with the following mutations: T82I, K10E, K34E, E156G, 181*, S38, H130, V152, R174, and T100; and compared with the sequence shown in SEQ ID NO:1, the corresponding nucleotide sequence of the mutant protein has at least 95% sequence identity. 
     
     
         2 . A High-efficiency Base Editor, comprising the mutant protein of activation-induced cytidine deaminase as claimed in  claim 1 , a DNA-specific binding protein and a nuclear localization signal, the mutant protein of activation-induced cytidine deaminase and the DNA-specific binding protein are sequentially liked via a linking sequence, and the nuclear localization signal is located at the C-terminus of the High-efficiency Base Editor. 
     
     
         3 . The High-efficiency Base Editor of  claim 2 , wherein the DNA-specific binding protein is a homing endonuclease; the corresponding nucleotide sequence of the DNA-specific binding protein is shown in SEQ ID NO: 2. 
     
     
         4 . The High-efficiency Base Editor of  claim 2 , further comprising a UGI protein domain, the UGI protein domain is located after the DNA-specific binding protein and before the nuclear localization signal. 
     
     
         5 . The High-efficiency Base Editor of  claim 3 , further comprising a UGI protein domain, the UGI protein domain is located after the DNA-specific binding protein and before the nuclear localization signal. 
     
     
         6 . The High-efficiency Base Editor of  claim 2 , wherein, it is suitable for yeast system, and its corresponding nucleotide sequence is shown in SEQ ID NO: 3;
 it is suitable for  Drosophila  system, and its corresponding nucleotide sequence is shown in SEQ ID NO: 4; or   it is suitable for zebrafish and mouse systems, and its corresponding nucleotide sequence is shown in SEQ ID NO:5.   
     
     
         7 . A single-base locus-directed editing system, comprising the single-base locus-directed editing protein of  claim 3  and a target Hyper Mutation Fragment. 
     
     
         8 . A single-base locus-directed editing system, comprising the single-base locus-directed editing protein of  claim 4  and a target Hyper Mutation Fragment. 
     
     
         9 . A single-base locus-directed editing system, comprising the single-base locus-directed editing protein of  claim 5  and a target Hyper Mutation Fragment. 
     
     
         10 . A single-base locus-directed editing system, comprising the single-base locus-directed editing protein of  claim 6  and a target Hyper Mutation Fragment. 
     
     
         11 . The single-base locus-directed editing system according to  claim 7 , wherein the target Hyper Mutation Fragment includes the nucleotide sequence as shown in SEQ ID NO: 6 and SEQ ID NO: 7. 
     
     
         12 . The single-base locus-directed editing system according to  claim 8 , wherein the target Hyper Mutation Fragment includes the nucleotide sequence as shown in SEQ ID NO: 6 and SEQ ID NO: 7. 
     
     
         13 . The single-base locus-directed editing system according to  claim 9 , wherein the target Hyper Mutation Fragment includes the nucleotide sequence as shown in SEQ ID NO: 6 and SEQ ID NO: 7. 
     
     
         14 . The single-base locus-directed editing system according to  claim 10 , wherein the target Hyper Mutation Fragment includes the nucleotide sequence as shown in SEQ ID NO: 6 and SEQ ID NO: 7. 
     
     
         15 . A gene editing method, comprising using the single-base locus-directed editing system of  claim 7 . 
     
     
         16 . A gene editing method, comprising using the single-base locus-directed editing system of  claim 8 . 
     
     
         17 . A gene editing method, comprising using the single-base locus-directed editing system of  claim 10 . 
     
     
         18 . A gene editing method, comprising using the single-base locus-directed editing system of  claim 11 . 
     
     
         19 . A gene editing method, comprising using the single-base locus-directed editing system of  claim 12 . 
     
     
         20 . A gene editing method, comprising using the single-base locus-directed editing system of  claim 14 .