Department of Bioregulation and Cellular Response, Graduate School of Medicine, Osaka University, Suita, Osaka, 565-0871, Japan.
Department of Genome Medicine, Tokyo Metropolitan Institute of Medical Science, Tokyo, 156-0057, Japan.
Nat Commun. 2023 Sep 15;14(1):5607. doi: 10.1038/s41467-023-41048-5.
CRISPR/Cas9-mediated gene editing has great potential utility for treating genetic diseases. However, its therapeutic applications are limited by unintended genomic alterations arising from DNA double-strand breaks and random integration of exogenous DNA. In this study, we propose NICER, a method for correcting heterozygous mutations that employs multiple nicks (MNs) induced by Cas9 nickase and a homologous chromosome as an endogenous repair template. Although a single nick near the mutation site rarely leads to successful gene correction, additional nicks on homologous chromosomes strongly enhance gene correction efficiency via interhomolog homologous recombination (IH-HR). This process partially depends on BRCA1 and BRCA2, suggesting the existence of several distinct pathways for MN-induced IH-HR. According to a genomic analysis, NICER rarely induces unintended genomic alterations. Furthermore, NICER restores the expression of disease-causing genes in cells derived from genetic diseases with compound heterozygous mutations. Overall, NICER provides a precise strategy for gene correction.
CRISPR/Cas9 介导的基因编辑在治疗遗传疾病方面具有巨大的潜在应用价值。然而，其治疗应用受到由 DNA 双链断裂和外源 DNA 随机整合引起的意外基因组改变的限制。在这项研究中，我们提出了 NICER 方法，该方法通过 Cas9 切口酶诱导的多个切口 (MNs) 和同源染色体作为内源性修复模板来纠正杂合突变。虽然突变位点附近的单个切口很少导致成功的基因校正，但同源染色体上的额外切口通过同源重组 (IH-HR) 强烈增强基因校正效率。这个过程部分依赖于 BRCA1 和 BRCA2，表明 MN 诱导的 IH-HR 存在几种不同的途径。根据基因组分析，NICER 很少引起意外的基因组改变。此外，NICER 恢复了具有复合杂合突变的遗传疾病来源的细胞中致病基因的表达。总的来说，NICER 提供了一种精确的基因校正策略。
