%0 English Abstract
%T [The Development of SpCas9 Variants with High Specificity and Efficiency Based on the HH Theory].
%A Wang GH
%A Wang CM
%A Wu XJ
%A Chu T
%A Huang DW
%A Li J
%J Mol Biol (Mosk)
%V 58
%N 1
%D 2024 Jan-Feb
%M 38943587
暂无%R 10.31857/S0026898424010158, EDN: NTCLJI
%X 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.