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.
摘要：
化脓性链球菌Cas9（SpCas9）是基因编辑中最流行的工具；然而，脱靶诱变是其应用的最大障碍之一。在我们之前的研究中,我们提出了HH理论，其中指出，sgRNA/DNA杂交体（杂交体）挤出诱导的杂交体与REC3/HNH之间疏水相互作用的增强是裂解起始的关键因素。基于HH理论,我们分析了REC3结构域与杂种之间的相互作用，并获得了8个突变位点。我们设计了8个SpCas9变体（V1-V8），使用数字液滴PCR评估SpCas9诱导的人细胞中的DNAindel，并开发了高保真变体。因此，HH理论可用于进一步优化SpCas9介导的基因组编辑系统，并且所得的V3、V6、V7和V8SpCas9变体对于需要高精度基因组编辑的应用可能是有价值的。
