The Development of SpCas9 Variants with High Specificity and Efficiency Based on the HH Theory

Wang, G. H.1; Wang, C. M.2,3; Wu, X. J.4; Chu, T.5; Huang, D. W.6; Li, J.7

2023-11-01

10.1134/S0026893324010187

MOLECULAR BIOLOGY   影响因子和分区

0026-8933

1608-3245

Streptococcus pyogenes Cas9 (SpCas9) is the most popular tool in gene editing; however, off-target mutagenesis is one of the biggest impediments in its application. In our previous study, we proposed the HH theory, which states that sgRNA/DNA hybrid (hybrid) extrusion-induced enhancement of hydrophobic interactions between the hybrid and REC3/HNH is a key factor in cleavage initiation. Based on the HH theory, we analyzed the interactions between the REC3 domain and hybrid and obtained 8 mutant sites. We designed 8 SpCas9 variants (V1-V8), used digital droplet PCR to assess SpCas9-induced DNA indels in human cells, and developed high-fidelity variants. Thus, the HH theory may be employed to further optimize SpCas9-mediated genome editing systems, and the resultant V3, V6, V7, and V8 SpCas9 variants may be valuable for applications requiring high-precision genome editing.

SpCas9 HH theory variants off-target cleavage efficiency optimization

SCI

英语

其他

Characteristic Innovation Project in General Colleges and Universities of Guangdong Province[2022KTSCX230] ; National Natural Science Foundation of China[81601654] ; Natural Science Foundation of Guangdong Province, China["2014A030310090","2016A030313578"] ; Medical Scientific Research Foundation of Guangdong Province, China[A2015207] ; Pearl River S&T Nova Program of Guangzhou[201806010037] ; Medical Science and Technology Research Foundation of Guangdong Province[A2023283] ; Nanshan District Science and Technology Plan Project[NS2022077]

Biochemistry & Molecular Biology

Biochemistry & Molecular Biology

WOS:001104718700001

PLEIADES PUBLISHING INC

MOLECULAR BIOLOGY & GENETICS

Web of Science

1.Guangdong Ind Polytech, Sch Food & Biotechnol, Guangzhou 510300, Peoples R China
2.Southern Med Univ, Nanfang Hosp, Dept Pharm, Guangzhou 510515, Peoples R China
3.Southern Univ Sci & Technol Hosp, Dept Pharm, Shenzhen 518000, Peoples R China
4.Univ North Carolina Chapel Hill, Lineberger Comprehens Canc Ctr, Chapel Hill, NC 27517 USA
5.Sangon Biotech, Shanghai 201611, Peoples R China
6.Jinhua Polytech, Pharmaceut & Mat Engn Sch, Jinhua 321007, Peoples R China
7.Southern Med Univ, Sch Basic Med Sci, Dept Histol & Embryol, Guangzhou 510515, Peoples R China

Wang, G. H.,Wang, C. M.,Wu, X. J.,et al. The Development of SpCas9 Variants with High Specificity and Efficiency Based on the HH Theory[J]. MOLECULAR BIOLOGY,2023.

Wang, G. H.,Wang, C. M.,Wu, X. J.,Chu, T.,Huang, D. W.,&Li, J..(2023).The Development of SpCas9 Variants with High Specificity and Efficiency Based on the HH Theory.MOLECULAR BIOLOGY.

Wang, G. H.,et al."The Development of SpCas9 Variants with High Specificity and Efficiency Based on the HH Theory".MOLECULAR BIOLOGY (2023).