Pooled genetic screening with CRISPR–Cas9 has enabled genome-wide, high-resolution mapping of genes to phenotypes, but assessing the effect of a given genetic perturbation requires evaluation of each single guide RNA (sgRNA) in hundreds of cells to counter stochastic genetic drift and obtain robust results. However, resolution is limited in complex, heterogeneous models, such as organoids or tumors transplanted into mice, because achieving sufficient representation requires impractical scaling. This is due to bottleneck effects and biological heterogeneity of cell populations. Here we introduce CRISPR-StAR, a screening method that uses internal controls generated by activating sgRNAs in only half the progeny of each cell subsequent to re-expansion of the cell clone. Our method overcomes both intrinsic and extrinsic heterogeneity as well as genetic drift in bottlenecks by generating clonal, single-cell-derived intrinsic controls. We use CRISPR-StAR to identify in-vivo-specific genetic dependencies in a genome-wide screen in mouse melanoma. Benchmarking against conventional screening demonstrates the improved data quality provided by this technology.

使用 CRISPR-Cas9 进行混合遗传筛选已经实现了基因到表型的全基因组高分辨率定位，但评估给定遗传扰动的影响需要评估数百个细胞中的每个单一向导 RNA （sgRNA），以对抗随机遗传漂变并获得可靠的结果。然而，在复杂的异质模型中，分辨率是有限的，例如移植到小鼠体内的类器官或肿瘤，因为要获得足够的表示需要不切实际的缩放。这是由于细胞群的瓶颈效应和生物学异质性。在这里，我们介绍了 CRISPR-StAR，这是一种筛选方法，它使用在细胞克隆重新扩增后仅在每个细胞的一半后代中激活 sgRNA 产生的内部对照。我们的方法通过生成克隆、单细胞衍生的内在对照来克服内在和外在异质性以及瓶颈中的遗传漂变。我们使用 CRISPR-StAR 在小鼠黑色素瘤的全基因组筛选中鉴定体内特异性遗传依赖性。与传统筛查的基准分析表明，该技术提高了数据质量。