US 12,129,479 B2
Genome editing method
Caixia Gao, Beijing (CN); Huawei Zhang, Beijing (CN); and Dingbo Zhang, Beijing (CN)
Assigned to SUZHOU QI BIODESIGN BIOTECHNOLOGY COMPANY LIMITED, Jiangsu (CN)
Filed by SUZHOU QI BIODESIGN BIOTECHNOLOGY COMPANY LIMITED, Suzhou (CN)
Filed on Apr. 3, 2023, as Appl. No. 18/130,300.
Application 18/130,300 is a continuation of application No. 16/487,024, abandoned, previously published as PCT/CN2018/076949, filed on Feb. 22, 2018.
Claims priority of application No. 201710089494.9 (CN), filed on Feb. 20, 2017.
Prior Publication US 2024/0011052 A1, Jan. 11, 2024
Int. Cl. C12N 15/90 (2006.01); A01H 1/06 (2006.01); C12N 9/22 (2006.01); C12N 15/01 (2006.01); C12N 15/113 (2010.01); C12N 15/82 (2006.01)
CPC C12N 15/902 (2013.01) [C12N 9/22 (2013.01); C12N 15/113 (2013.01); C12N 15/8213 (2013.01); C12N 2310/20 (2017.05); C12N 2310/3519 (2013.01)] 7 Claims
 
1. A method for genetically modifying a cell, comprising:
introducing into the cell a genome editing system for site-directed modification of a target sequence, wherein 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,
wherein the genome editing system 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 Cas9 nuclease variant comprises the amino acid sequence as set forth in SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6; wherein the 5′ end of the guide RNA is linked to the 3′ end of the tRNA, and the guide RNA is 20 nt in length; and wherein the fusion of guide RNA and the tRNA 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.