Department of Bioregulation and Cellular Response, Graduate School of Medicine, Osaka University, Suita, Osaka 565-0871, Japan.
Mouse Genome Engineering Core Facility, Vice Chancellor for Research Office, University of Nebraska Medical Center, Omaha, Nebraska 68198, USA.
Genome Res. 2018 Feb;28(2):223-230. doi: 10.1101/gr.226027.117. Epub 2017 Dec 22.
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.
CRISPR/Cas9 在靶标位点产生 DNA 双链断裂 (DSBs)，当与供体 DNA 模板共递用时，是编辑基因组的有力工具。然而，DSBs 是最具危害性的 DNA 损伤类型，经常通过诱变的非同源末端连接导致非预期的核苷酸插入/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中进行单个切口 (SNGD) 的组合促进了通过基因编辑的有效核苷酸替换。单独切口靶基因不能促进有效的基因编辑。然而，供体质粒骨架中的额外切口显著提高了基因编辑效率。SNGD 介导的基因编辑导致的 indel 频率明显低于 DSB 介导的方法。我们还表明，SNGD 促进了人细胞内内源基因座的基因编辑。从机制上讲，SNGD 介导的基因编辑需要靶基因和修复模板之间的长序列同源性，但不需要 CtIP、RAD51 或 RAD52。因此，人们认为非规范同源定向修复调节 SNGD 介导的基因编辑。总之，SNGD 促进了精确和高效的基因编辑，可能是开发新型基因治疗方法的有前途的策略。
