CRISPR-Cas9 genome editing in human cells occurs via the Fanconi anemia pathway
CRISPR-Cas9 genome editing in human cells occurs via the Fanconi anemia pathway
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DOI：
10.1038/s41588-018-0174-0
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发表时间：
2018-08-01
期刊：
影响因子：
30.8
通讯作者：
Corn, Jacob E.
中科院分区：
文献类型：
--
作者：
Richardson, Chris D.;Kazane, Katelynn R.;Corn, Jacob E.
CRISPR-Cas 基因组编辑可产生靶向 DNA 双链断裂 (DSB)，并通过细胞修复途径进行处理，包括通过单链模板修复 (SSTR) 掺入外源 DNA。为了确定人类细胞中 SSTR 的遗传基础，我们开发了一种耦合抑制切割系统，能够在数千个个体基因敲低的情况下询问多种编辑结果。我们发现人类 Cas9 诱导的 SSTR 需要范可尼贫血 (FA) 途径，该途径通常与链间交联修复有关。 FA 途径不会直接影响容易出错的非同源末端连接，而是将修复转向 SSTR。此外，FANCD2 蛋白定位于 Cas9 诱导的 DSB，表明在调节基因组编辑中具有直接作用。由于 FA 本身就是一种遗传性疾病，这些数据意味着患者基因型和/或转录组可能会影响基因编辑治疗的有效性，并且偏向 FA 修复途径的治疗可能具有治疗价值。
CRISPR-Cas genome editing creates targeted DNA double-strand breaks (DSBs) that are processed by cellular repair pathways, including the incorporation of exogenous DNA via single-strand template repair (SSTR). To determine the genetic basis of SSTR in human cells, we developed a coupled inhibition-cutting system capable of interrogating multiple editing outcomes in the context of thousands of individual gene knockdowns. We found that human Cas9-induced SSTR requires the Fanconi anemia ( FA) pathway, which is normally implicated in interstrand cross-link repair. The FA pathway does not directly impact error-prone, non-homologous end joining, but instead diverts repair toward SSTR. Furthermore, FANCD2 protein localizes to Cas9-induced DSBs, indicating a direct role in regulating genome editing. Since FA is itself a genetic disease, these data imply that patient genotype and/or transcriptome may impact the effectiveness of gene editing treatments and that treatments biased toward FA repair pathways could have therapeutic value.