CRISPR-Cas9已成为基因组编辑的强大工具。然而,Cas9基因组编辑面临挑战包括低效率和脱靶效应。这里,我们报道了与RAD51联合治疗,RAD51是同源重组的关键因素,和SCR7,一种DNA连接酶IV小分子抑制剂,增强CRISPR-Cas9介导的人胚肾293T和人诱导多能干细胞的基因组编辑效率,通过细胞透射电子显微镜和功能分析证实。首先,我们的发现揭示了RAD51在同源重组(HR)介导的DNA修复过程中的关键作用。外源性RAD51水平的升高通过单链DNA缺口修复过程促进复制后的步骤,确保完成DNA复制。第二,使用一体化CRISPR-Cas9-RAD51系统,高表达的RAD51通过与SCR7协同激活基于HR的修复途径,提高了多个内源性基因敲入/敲除效率和插入/缺失(InDel)突变.Sanger测序显示RAD51-SCR7在多个基因组位点中的InDel突变比率中的不同结果。第三,RAD51-SCR7组合可以通过增强HR过程诱导有效的R环分辨率和DNA修复,这导致DNA复制停滞,因此有利于基于CRISPR-Cas9的稳定基因组编辑。我们的研究表明,通过RAD51和SCR7提高CRISPR-Cas9效率,在基因组编辑中具有广阔的应用前景,为生物技术和治疗提供了潜在的进步。
CRISPR-Cas9 has emerged as a powerful tool for genome editing. However, Cas9 genome editing faces challenges, including low efficiency and off-target effects. Here, we report that combined treatment with RAD51, a key factor in homologous recombination, and SCR7, a DNA ligase IV small-molecule inhibitor, enhances CRISPR-Cas9-mediated genome-editing efficiency in human embryonic kidney 293T and human induced pluripotent stem cells, as confirmed by cyro- transmission electron microscopy and functional analyses. First, our findings reveal the crucial role of RAD51 in homologous recombination (HR)-mediated DNA repair process. Elevated levels of exogenous RAD51 promote a post-replication step via single-strand DNA gap repair process, ensuring the completion of DNA replication. Second, using the all-in-one CRISPR-Cas9-RAD51 system, highly expressed RAD51 improved the multiple endogenous gene knockin/knockout efficiency and insertion/deletion (InDel) mutation by activating the HR-based repair pathway in concert with SCR7. Sanger sequencing shows distinct outcomes for RAD51-SCR7 in the ratio of InDel mutations in multiple genome sites. Third, RAD51-SCR7 combination can induce efficient R-loop resolution and DNA repair by enhanced HR process, which leads to DNA replication stalling and thus is advantageous to CRISPR-Cas9-based stable genome editing. Our study suggests promising applications in genome editing by enhancing CRISPR-Cas9 efficiency through RAD51 and SCR7, offering potential advancements in biotechnology and therapeutics.