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.

中文翻译：

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基于HH理论开发高特异性、高效率的SpCas9变体

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，该理论指出，sgRNA/DNA杂合体（hybrid）挤压诱导的杂合体与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结构域与杂交体之间的相互作用，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 评估人类细胞中 SpCas9 诱导的 DNA 插入缺失，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，所得的V3、V6、V7和V8 SpCas9变体对于需要高精度基因组编辑的应用可能有价值。