US 11,739,322 B2
Method for genome editing using a self-inactivating CRISPR nuclease
Caixia Gao, Beijing (CN); Huawei Zhang, Shandong Province (CN); and Shuai Jin, Beijing (CN)
Assigned to INSTITUTE OF GENETICS AND DEVELOPMENTAL BIOLOGY, CHINESE ACADEMY OF SCIENCES, Beijing (CN)
Appl. No. 16/966,538
Filed by INSTITUTE OF GENETICS AND DEVELOPMENTAL BIOLOGY, CHINESE ACADEMY OF SCIENCES, Beijing (CN)
PCT Filed Jan. 31, 2019, PCT No. PCT/CN2019/074088
§ 371(c)(1), (2) Date Jul. 31, 2020,
PCT Pub. No. WO2019/149239, PCT Pub. Date Aug. 8, 2019.
Claims priority of application No. 201810101165.6 (CN), filed on Feb. 1, 2018.
Prior Publication US 2021/0047639 A1, Feb. 18, 2021
Int. Cl. C12N 15/64 (2006.01); C12N 15/11 (2006.01); C12N 9/22 (2006.01); C12N 15/90 (2006.01)
CPC C12N 15/11 (2013.01) [C12N 9/22 (2013.01); C12N 15/902 (2013.01); C12N 2310/20 (2017.05); C12N 2510/02 (2013.01); C12N 2800/80 (2013.01)] 9 Claims
 
1. A genome editing system for site-directed modification of at least one genomic target sequence in the genome of a cell, comprising:
1) an expression construct comprising a coding sequence of a gRNA targeting the at least one genomic target sequence;
2) an expression construct comprising a coding sequence of a CRISPR nuclease; and
3) an expression construct comprising coding sequence for a gRNA targeting a target sequence within the coding sequence of the CRISPR nuclease,
wherein, upon introduction into the cell, said gRNA targeting the at least one genomic target sequence directs the CRISPR nuclease to said at least one genomic target sequence and results in one or more mutations in the genomic target sequence, and the gRNA targeting a target sequence within the coding sequence of the CRISPR nuclease directs the CRISPR nuclease to said target sequence within the coding sequence of the CRISPR nuclease and results in an inactivating mutation of the CRISPR nuclease,
wherein the CRISPR nuclease is a single-base editing CRISPR nuclease comprising the amino acid sequence set forth in SEQ ID NO: 1,
wherein the nucleotide sequence encoding the single-base editing CRISPR nuclease is set forth in SEQ ID NO: 2, and
wherein the target sequence within the coding sequence of the CRISPR nuclease is set forth in SEQ ID NO: 3.