Department of Genetics and Development, Herbert Irving Comprehensive Cancer Center, Columbia University Irving Medical Center, New York, NY, 10032, USA.
Naomi Berrie Diabetes Center and Department of Pediatrics, Columbia University Irving Medical Center, New York, NY, 10032, USA.
Nat Commun. 2019 Jul 30;10(1):3395. doi: 10.1038/s41467-019-11105-z.
Precise editing of genomic DNA can be achieved upon repair of CRISPR-induced DNA double-stranded breaks (DSBs) by homology-directed repair (HDR). However, the efficiency of this process is limited by DSB repair pathways competing with HDR, such as non-homologous end joining (NHEJ). Here we individually express in human cells 204 open reading frames involved in the DNA damage response (DDR) and determine their impact on CRISPR-mediated HDR. From these studies, we identify RAD18 as a stimulator of CRISPR-mediated HDR. By defining the RAD18 domains required to promote HDR, we derive an enhanced RAD18 variant (e18) that stimulates CRISPR-mediated HDR in multiple human cell types, including embryonic stem cells. Mechanistically, e18 induces HDR by suppressing the localization of the NHEJ-promoting factor 53BP1 to DSBs. Altogether, this study identifies e18 as an enhancer of CRISPR-mediated HDR and highlights the promise of engineering DDR factors to augment the efficiency of precision genome editing.
通过同源定向修复 (HDR) 修复 CRISPR 诱导的 DNA 双链断裂 (DSB)，可以实现基因组 DNA 的精确编辑。然而，这个过程的效率受到 DSB 修复途径与 HDR 竞争的限制，例如非同源末端连接 (NHEJ)。在这里，我们在人类细胞中分别表达 204 个涉及 DNA 损伤反应 (DDR) 的开放阅读框，并确定它们对 CRISPR 介导的 HDR 的影响。通过这些研究，我们确定 RAD18 是 CRISPR 介导的 HDR 的促进剂。通过定义促进 HDR 所需的 RAD18 结构域，我们推导出一种增强型 RAD18 变体 (e18)，它可以刺激多种人类细胞类型（包括胚胎干细胞）中的 CRISPR 介导的 HDR。从机制上讲，e18 通过抑制促进 NHEJ 的因子 53BP1 定位到 DSB 来诱导 HDR。总之，这项研究确定了 e18 是 CRISPR 介导的 HDR 的增强剂，并强调了工程 DDR 因子以提高精确基因组编辑效率的潜力。
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