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- Title
The Development of SpCas9 Variants with High Specificity and Efficiency Based on the HH Theory.
- Authors
Wang, G. H.; Wang, C. M.; Wu, X. J.; Chu, T.; Huang, D. W.; Li, J.
- Abstract
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.
- Subjects
STREPTOCOCCUS pyogenes; HYDROPHOBIC interactions; HUMAN DNA; GENOME editing; MUTAGENESIS
- Publication
Molecular Biology, 2024, Vol 58, Issue 1, p133
- ISSN
0026-8933
- Publication type
Article
- DOI
10.1134/S0026893324010187