Genome editing method

Abstract

The present invention relates to the field of genetic engineering. In particular, the present invention relates to a genome editing method with high efficiency and high specificity. More specifically, the present invention relates to a method for increasing the efficiency of site-directed modification of a target sequence in a genome of an organism by a high-specificity Cas9 nuclease variant.

1 . A genome editing system for site-directed modification of a target sequence in the genome of a cell, which comprises at least one selected from the following i) to iii):
 i) a Cas9 nuclease variant, and an expression construct comprising a nucleotide sequence encoding a tRNA-guide RNA fusion;   ii) an expression construct comprising a nucleotide sequence encoding a Cas9 nuclease variant, and an expression construct comprising a nucleotide sequence encoding a tRNA-guide RNA fusion; and   iii) an expression construct comprising a nucleotide sequence encoding a Cas9 nuclease variant and a nucleotide sequence encoding a tRNA-guide RNA fusion;   wherein the Cas9 nuclease variant has higher specificity as compared with the wild-type Cas9 nuclease,   wherein the 5′ end of the guide RNA is linked to the 3′ end of the tRNA, wherein the fusion is cleaved at the 5′ end of the guide RNA after being transcribed in the cell, thereby forming a guide RNA that does not carry an extra nucleotide at the 5′ end.   
     
     
         2 . A genome editing system for site-directed modification of a target sequence in the genome of a cell, which comprises at least one selected from the following i) to iii):
 i) a Cas9 nuclease variant, and an expression construct comprising a nucleotide sequence encoding a ribozyme-guide RNA fusion;   ii) an expression construct comprising a nucleotide sequence encoding a Cas9 nuclease variant, and an expression construct comprising a nucleotide sequence encoding a ribozyme-guide RNA fusion; and   iii) an expression construct comprising a nucleotide sequence encoding a Cas9 nuclease variant and a nucleotide sequence encoding a ribozyme-guide RNA fusion;   wherein the Cas9 nuclease variant has higher specificity as compared with the wild-type Cas9 nuclease,   wherein the 5′ end of the guide RNA is linked to the 3′ end of a first ribozyme,   wherein the first ribozyme is designed to cleave the fusion at the 5′ end of the guide RNA, thereby forming a guide RNA that does not carry extra nucleotide at the 5′ end.   
     
     
         3 . The system of  claim 1 , wherein the tRNA and the cell to be modified are derived from a same species. 
     
     
         4 . The system of  claim 1 , wherein the tRNA is encoded by a sequence as shown in SEQ ID NO:1. 
     
     
         5 . The system of  claim 1 , wherein the Cas9 nuclease variant is a variant of SEQ ID NO:2 and comprises an amino acid substitution at position 855 of SEQ ID NO:2, for example, the amino acid substitution is K855A. 
     
     
         6 . The system of  claim 1 , wherein the Cas9 nuclease variant is a variant of the SEQ ID NO:2 and comprises amino acid substitutions at positions 810, 1003 and 1060 of SEQ ID NO:2, for example, the amino acid substitutions are K810A, K1003A and R1060A. 
     
     
         7 . The system of  claim 1 , wherein the Cas9 nuclease variant is a variant of the SEQ ID NO:2 and comprises amino acid substitutions at positions 848, 1003 and 1060 of SEQ ID NO:2, for example, the amino acid substitutions are K848A, K1003A and R1060A. 
     
     
         8 . The system of  claim 1 , wherein the Cas9 nuclease variant is a variant of the SEQ ID NO:2 and comprises amino acid substitutions at positions 611, 695 and 926 of SEQ ID NO:2, for example, the amino acid substitutions are R611A, Q695A and Q926A. 
     
     
         9 . The system of  claim 1 , wherein the Cas9 nuclease variant is a variant of the SEQ ID NO:2 and comprises amino acid substitutions at positions 497, 611, 695 and 926 of SEQ ID NO:2, for example, the amino acid substitutions are N497A, R611A, Q695A and Q926A. 
     
     
         10 . The system of  claim 1 , wherein the Cas9 nuclease variant comprises an amino acid sequence as shown in SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6. 
     
     
         11 . The system of  claim 1 , wherein the nucleotide sequence encoding the Cas9 nuclease variant is codon-optimized for the organism from which the cell to be modified is derived. 
     
     
         12 . The system of  claim 1 , wherein the guide RNA is a single guide RNA (sgRNA). 
     
     
         13 . A method for genetically modifying a cell, comprising: introducing the system of  claim 1  to the cell, and thereby the Cas9 nuclease variant is targeted to the target sequence in the genome of the cell by the guide RNA, and results in substitution, deletion and/or addition of one or more nucleotides in the target sequence. 
     
     
         14 . The method of  claim 13 , wherein the cell is derived from mammals. 
     
     
         15 . The method of  claim 13 , wherein the system is introduced into the cell by a method selected from: calcium phosphate transfection, protoplast fusion, electroporation, liposome transfection, microinjection, viral infection (such as a baculovirus, a vaccinia virus, an adenovirus and other viruses), particle bombardment, PEG-mediated protoplast transformation and agrobacterium-mediated transformation. 
     
     
         16 . The method of  claim 14 , wherein the mammal is a human, a mouse, a rat, a monkey, a dog, a pig, a sheep, a cow or a cat, wherein the poultry is a chicken, a duck or a goose, and wherein the plant is rice, maize, wheat, sorghum, barley, soybean, peanut or  Arabidopsis thaliana.