Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, ON M5G 1X5, Canada.
Department of Molecular Genetics, University of Toronto, 1 King's College Circle, Toronto, ON M5S 1A8, Canada.
STAR Protoc. 2021 Feb 5;2(1):100321. doi: 10.1016/j.xpro.2021.100321. eCollection 2021 Mar 19.
CRISPR-based genetic screens revolutionized our ability to genetically probe cell biology. We present a protocol to conduct genome-scale chemogenomic dropout CRISPR screens in the human RPE1-hTERT p53 cell line. We use the TKOv3 library, which contains 70,948 sgRNAs targeting 18,053 genes. Here, we describe how to set up the screen, the reagents required, and how to sequence and analyze the results. This protocol can be customized for other libraries, cell lines, and sequencing instruments. For complete details on the use and execution of this protocol, please refer to Olivieri et al. (2020).
基于 CRISPR 的遗传筛选技术极大地提高了我们在遗传水平上研究细胞生物学的能力。我们介绍了一种在人视网膜色素上皮 1 细胞系（RPE1-hTERT p53）中进行全基因组化学基因组 CRISPR 筛选的方案。我们使用了 TKOv3 文库，其中包含 70948 个靶向 18053 个基因的 sgRNA。在此，我们将描述如何设置筛选、所需的试剂以及如何对结果进行测序和分析。此方案可以针对其他文库、细胞系和测序仪器进行定制。有关此方案的使用和执行的完整详细信息，请参阅 Olivieri 等人（2020 年）。
