Parallel CRISPR-Cas9 screens clarify impacts of p53 on screen performance
Parallel CRISPR-Cas9 screens clarify impacts of p53 on screen performance
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DOI：
10.7554/elife.55325
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发表时间：
2020-05-22
期刊：
影响因子：
7.7
通讯作者：
Jackson, Stephen P.
中科院分区：
文献类型：
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作者：
Bowden, Anne Ramsay;Morales-Juarez, David A.;Jackson, Stephen P.
CRISPR-Cas9基因组工程使高通量功能基因组筛选发生了革命性的变化。然而，最近的工作引起了人们对使用TP53野生型人类细胞的CRISPR-Cas9筛查性能的担忧，因为p53介导的DNA损伤反应(DDR)限制了生成可编辑细胞的效率。为了直接评估细胞p53状态对CRISPR-Cas9筛选性能的影响，我们使用针对852个DDR相关基因的聚焦双引导RNA文库，在野生型和TP53基因敲除的人视网膜色素上皮细胞中进行了平行的CRISPR-Cas9筛选。我们的工作表明，尽管功能性P53状态对显著耗尽的基因的识别具有负面影响，但优化的屏幕设计仍然可以实现稳健的屏幕性能。通过对我们自己和发表的筛查数据的分析，我们强调了在野生型和p53缺陷细胞中成功筛查的关键因素。
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 TP53 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 TP53 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.