Wellcome/Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge, United Kingdom.
Elife. 2020 May 22;9:e55325. doi: 10.7554/eLife.55325.
CRISPR-Cas9 genome engineering has revolutionised high-throughput functional genomic screens. However, recent work has raised concerns regarding the performance of CRISPR-Cas9 screens using wild-type human cells due to a p53-mediated DNA damage response (DDR) limiting the efficiency of generating viable edited cells. To directly assess the impact of cellular p53 status on CRISPR-Cas9 screen performance, we carried out parallel CRISPR-Cas9 screens in wild-type and knockout human retinal pigment epithelial cells using a focused dual guide RNA library targeting 852 DDR-associated genes. Our work demonstrates that although functional p53 status negatively affects identification of significantly depleted genes, optimal screen design can nevertheless enable robust screen performance. Through analysis of our own and published screen data, we highlight key factors for successful screens in both wild-type and p53-deficient cells.
CRISPR-Cas9 基因组工程已经彻底改变了高通量功能基因组筛选。然而，最近的工作对使用野生型人类细胞进行 CRISPR-Cas9 筛选的性能提出了担忧，这是由于 p53 介导的 DNA 损伤反应（DDR）限制了生成有活力的编辑细胞的效率。为了直接评估细胞 p53 状态对 CRISPR-Cas9 筛选性能的影响，我们使用靶向 852 个 DDR 相关基因的聚焦双向导 RNA 文库，在野生型和 knockout 人视网膜色素上皮细胞中进行平行 CRISPR-Cas9 筛选。我们的工作表明，尽管功能性 p53 状态会对显著耗尽基因的鉴定产生负面影响，但优化的筛选设计仍然可以实现稳健的筛选性能。通过分析我们自己和已发表的筛选数据，我们强调了在野生型和 p53 缺陷细胞中成功筛选的关键因素。
