Innovative Genomics Institute, University of California, Berkeley, CA, 94703, USA.
Department of Molecular and Cell Biology, University of California, Berkeley, CA, 94703, USA.
Nat Commun. 2020 Apr 30;11(1):2109. doi: 10.1038/s41467-020-15845-1.
Repair of double strand DNA breaks (DSBs) can result in gene disruption or gene modification via homology directed repair (HDR) from donor DNA. Altering cellular responses to DSBs may rebalance editing outcomes towards HDR and away from other repair outcomes. Here, we utilize a pooled CRISPR screen to define host cell involvement in HDR between a Cas9 DSB and a plasmid double stranded donor DNA (dsDonor). We find that the Fanconi Anemia (FA) pathway is required for dsDonor HDR and that other genes act to repress HDR. Small molecule inhibition of one of these repressors, CDC7, by XL413 and other inhibitors increases the efficiency of HDR by up to 3.5 fold in many contexts, including primary T cells. XL413 stimulates HDR during a reversible slowing of S-phase that is unexplored for Cas9-induced HDR. We anticipate that XL413 and other such rationally developed inhibitors will be useful tools for gene modification.
双链 DNA 断裂 (DSBs) 的修复可能会导致基因中断或基因修饰，这是通过同源定向修复 (HDR) 从供体 DNA 实现的。改变细胞对 DSBs 的反应可以使编辑结果向 HDR 倾斜，减少其他修复结果。在这里，我们利用一个 pooled CRISPR 筛选来定义细胞在 Cas9 DSB 和质粒双链供体 DNA (dsDonor) 之间 HDR 中的宿主细胞参与。我们发现，范可尼贫血 (FA) 途径是 dsDonor HDR 所必需的，而其他基因则起到抑制 HDR 的作用。CDC7 等这些抑制剂的小分子抑制可以在许多情况下，包括原代 T 细胞中，将 HDR 的效率提高多达 3.5 倍。XL413 通过可逆减缓 S 期来刺激 HDR，而 Cas9 诱导的 HDR 在此期间并没有得到探索。我们预计，XL413 和其他类似的合理开发的抑制剂将成为基因修饰的有用工具。
