CRISPR/Cas9, which generates DNA double-strand breaks (DSBs) at target loci, is a powerful tool for editing genomes when codelivered with a donor DNA template. However, DSBs, which are the most deleterious type of DNA damage, often result in unintended nucleotide insertions/deletions (indels) via mutagenic nonhomologous end joining. We developed a strategy for precise gene editing that does not generate DSBs. We show that a combination of single nicks in the target gene and donor plasmid (SNGD) using Cas9D10A nickase promotes efficient nucleotide substitution by gene editing. Nicking the target gene alone did not facilitate efficient gene editing. However, an additional nick in the donor plasmid backbone markedly improved the gene-editing efficiency. SNGD-mediated gene editing led to a markedly lower indel frequency than that by the DSB-mediated approach. We also show that SNGD promotes gene editing at endogenous loci in human cells. Mechanistically, SNGD-mediated gene editing requires long-sequence homology between the target gene and repair template, but does not require CtIP, RAD51, or RAD52. Thus, it is considered that noncanonical homology-directed repair regulates the SNGD-mediated gene editing. In summary, SNGD promotes precise and efficient gene editing and may be a promising strategy for the development of a novel gene therapy approach.

中文翻译：

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通过非规范同源性指导的修复，在基因组缺口附近进行精确而有效的核苷酸置换。

CRISPR / Cas9在目标基因座上产生DNA双链断裂（DSB），是在与供体DNA模板一起编码传递时编辑基因组的强大工具。但是，DSB是最有害的DNA损伤类型，通常会通过诱变的非同源末端连接而导致意外的核苷酸插入/缺失（indels）。我们开发了一种不产生DSB的精确基因编辑策略。我们表明，使用Cas9D10A切口酶在靶基因和供体质粒（SNGD）中的单个切口的组合促进了通过基因编辑的有效核苷酸取代。单单剪切靶基因并不能促进有效的基因编辑。但是，在供体质粒主链中的另一个缺口明显改善了基因编辑效率。SNGD介导的基因编辑比DSB介导的方法显着降低插入缺失频率。我们还显示，SNGD可以促进人类细胞内源性基因座的基因编辑。从机理上讲，SNGD介导的基因编辑需要靶基因与修复模板之间具有长序列同源性，但不需要CtIP，RAD51或RAD52。因此，认为非规范的同源性指导的修复调节了SNGD介导的基因编辑。总之，SNGD促进精确和有效的基因编辑，并且可能是开发新型基因治疗方法的有前途的策略。因此，认为非规范的同源性指导的修复调节了SNGD介导的基因编辑。总之，SNGD促进精确和有效的基因编辑，并且可能是开发新型基因治疗方法的有前途的策略。因此，认为非规范的同源性指导的修复调节了SNGD介导的基因编辑。总之，SNGD促进精确和有效的基因编辑，并且可能是开发新型基因治疗方法的有前途的策略。