Genetic Perturbation Platform, Broad Institute, Cambridge, MA 02142, USA.
Cancer Program, Broad Institute, Cambridge, MA 02142, USA.
Cell. 2021 Feb 18;184(4):1064-1080.e20. doi: 10.1016/j.cell.2021.01.012.
Understanding the functional consequences of single-nucleotide variants is critical to uncovering the genetic underpinnings of diseases, but technologies to characterize variants are limiting. Here, we leverage CRISPR-Cas9 cytosine base editors in pooled screens to scalably assay variants at endogenous loci in mammalian cells. We benchmark the performance of base editors in positive and negative selection screens, identifying known loss-of-function mutations in BRCA1 and BRCA2 with high precision. To demonstrate the utility of base editor screens to probe small molecule-protein interactions, we screen against BH3 mimetics and PARP inhibitors, identifying point mutations that confer drug sensitivity or resistance. We also create a library of single guide RNAs (sgRNAs) predicted to generate 52,034 ClinVar variants in 3,584 genes and conduct screens in the presence of cellular stressors, identifying loss-of-function variants in numerous DNA damage repair genes. We anticipate that this screening approach will be broadly useful to readily and scalably functionalize genetic variants.
理解单核苷酸变异的功能后果对于揭示疾病的遗传基础至关重要，但用于描述变异的技术却受到限制。在这里，我们利用 CRISPR-Cas9 胞嘧啶碱基编辑器在汇集的筛选中，在哺乳动物细胞中对内在基因座的变异进行规模化分析。我们对碱基编辑器在正选择和负选择筛选中的性能进行了基准测试，高精度地鉴定出 BRCA1 和 BRCA2 中的已知功能丧失突变。为了证明碱基编辑器筛选在探测小分子-蛋白质相互作用方面的效用，我们针对 BH3 模拟物和 PARP 抑制剂进行筛选，鉴定出赋予药物敏感性或耐药性的点突变。我们还创建了一个预测会在 3584 个基因中产生 52034 个 ClinVar 变异的单指导 RNA（sgRNA）文库，并在细胞应激条件下进行筛选，鉴定出许多 DNA 损伤修复基因中的功能丧失变异。我们预计这种筛选方法将广泛适用于简便、规模化地对遗传变异进行功能化。
