Girish Vishruth, Sheltzer Jason M
Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA.
Bio Protoc. 2020 Jul 20;10(14):e3682. doi: 10.21769/BioProtoc.3682.
The CRISPR/Cas9 system is a powerful tool for genome editing, wherein the RNA-guided nuclease Cas9 can be directed to introduce double-stranded breaks (DSBs) at a targeted locus. In mammalian cells, these DSBs are typically repaired through error-prone processes, resulting in insertions or deletions (indels) at the targeted locus. Researchers can use these Cas9-mediated lesions to probe the consequences of loss-of-function perturbations in genes of interest. Here, we describe an optimized protocol to identify specific genes required for cancer cell fitness through a CRISPR-mediated cellular competition assay. Identifying these genetic dependencies is of utmost importance, as they provide potential targets for anti-cancer drug development. This protocol provides researchers with a robust and scalable approach to investigate gene dependencies in a variety of cell lines and cancer types and to validate the results of high-throughput or whole-genome screens.
CRISPR/Cas9系统是一种强大的基因组编辑工具，其中RNA引导的核酸酶Cas9可被定向到目标位点引入双链断裂（DSB）。在哺乳动物细胞中，这些DSB通常通过易出错的过程进行修复，导致目标位点出现插入或缺失（indel）。研究人员可以利用这些Cas9介导的损伤来探究感兴趣基因功能丧失性扰动的后果。在此，我们描述了一种优化方案，通过CRISPR介导的细胞竞争试验来鉴定癌细胞适应性所需的特定基因。确定这些基因依赖性至关重要，因为它们为抗癌药物开发提供了潜在靶点。该方案为研究人员提供了一种强大且可扩展的方法，用于研究各种细胞系和癌症类型中的基因依赖性，并验证高通量或全基因组筛选的结果。
