CRISPR-StAR, a paradigm leveraging internal controls, empowers genetic screening in vivo (batch 1)

Pooled genetic screening with CRISPR/Cas9 has enabled genome-wide high-resolution assignment of genes to phenotypes. To assess the effect of a given genetic perturbation, each sgRNA must be evaluated in hundreds of cells to overcome stochastic genetic drift and obtain robust results. In complex models that display particularly high heterogeneity, such as organoids or tumors transplanted into mice however, sufficient representation typically requires unreasonable scaling, thus preventing genome-wide screens at high resolution. Here we present CRISPR-StAR, a screening paradigm that overcomes intrinsic and extrinsic heterogeneity as well as genetic drift in bottlenecks by leveraging internal controls generated through activating sgRNAs only in half of the progenies of each cell. We use CRISPR-StAR to reveal in vivo-specific genetic dependencies in a genome-wide screen in mouse melanoma. Benchmarking to a conventional screening setup highlights the improved data quality this technology delivers. We anticipate CRISPR-StAR to set a new standard for genetic screening in complex models, foremost in vivo. Overall design: To investigate the in vivo-specific dependencies in melanoma, we performed a genome-wide CRISPR/Cas9 screen, as well as smaller validation screens. The samples obtained from the screened in vivo tumors and in vitro cells were processed with NGS. And subsequently analysed using Model-based Analysis of Genome-wide CRISPR-Cas9 Knockout (MAGeCK). However, we performed analysis according the CRISPR-StAR analysis method. Genome-wide screen was performed in 2 batches.

基于CRISPR/Cas9的混合池遗传筛选已实现全基因组高分辨率的基因-表型关联注释。为评估特定遗传扰动的效应，需在数百个细胞中对每条单向导RNA（single guide RNA, sgRNA）进行评估，以克服随机遗传漂变并获得可靠结果。然而，在具有高度异质性的复杂模型（如类器官或移植入小鼠体内的肿瘤）中，要达到足够的细胞代表性通常需要不合理的样本规模扩张，从而无法开展高分辨率的全基因组筛选。本研究提出CRISPR-StAR技术，这是一种筛选范式，通过仅在每个细胞的半数子代中激活单向导RNA以生成内参，从而克服固有与外在异质性及瓶颈效应下的遗传漂变。我们利用CRISPR-StAR技术，在小鼠黑色素瘤的全基因组筛选中揭示了体内特异性的遗传依赖关系。通过与传统筛选方案进行基准对比，证实该技术可显著提升数据质量。我们预计CRISPR-StAR将为复杂模型（尤其是体内模型）中的遗传筛选树立新的标准。实验设计概述：为探究黑色素瘤的体内特异性遗传依赖关系，我们开展了全基因组CRISPR/Cas9筛选及小型验证筛选。从经筛选的体内肿瘤及体外培养细胞中获取的样本，通过下一代测序（Next-Generation Sequencing, NGS）进行处理，随后采用全基因组CRISPR-Cas9敲除模型分析工具（Model-based Analysis of Genome-wide CRISPR-Cas9 Knockout, MAGeCK）进行分析。但我们同时依据CRISPR-StAR专属分析流程开展了数据分析。全基因组筛选共分2批完成。