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Vol 58(2024) N 1 p. 133-146; DOI 10.1134/S0026893324010187  G.H. Wang1, C.M. Wang2,3, X.J. Wu4, T. Chu5, D.W. Huang6, J. Li7* The Development of SpCas9 Variants with High Specificity and Efficiency Based on the HH Theory 1College of Life and Health Sciences, Guangdong Industry Polytechnic, Guangzhou, 510300 China2Department of Pharmacy, Nanfang Hospital, Southern Medical University, Guangzhou, 510515 China 3Department of Pharmacy, Southern University of Science and Technology Hospital, Shenzhen, 518000 China 4Lineberger Comprehensive Cancer Center, the University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, 27517 USA 5Sangon Biotech, Shanghai, 201611 China 6Pharmaceutical and Material Engineering School, Jinhua Polytechnic, Jinhua, 321007 China 7Department of Histology and Embryology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, 510515 China Received - 2023-04-01; Revised - 2023-07-31; Accepted - 2023-08-15 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 |
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