CRISPR-StAR, a paradigm leveraging internal controls, empowers genetic screening in vivo (batch 2). CRISPR-StAR, a paradigm leveraging internal controls, empowers genetic screening in vivo (batch 2)

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的混合池遗传筛选技术，已实现全基因组范围内高分辨率的基因-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引导RNA（sgRNA, single guide RNA）进行评估，以克服随机遗传漂变并获得可靠的实验结果。然而，对于呈现高度异质性的复杂模型（如类器官或移植到小鼠体内的肿瘤），要获得足够的细胞代表性通常需要进行不合理的规模化扩增，这阻碍了高分辨率全基因组筛选的开展。本研究推出CRISPR-StAR技术，这是一种新型筛选范式：通过仅在每个细胞的半数子代中激活单引导RNA以生成内参对照，从而克服固有与外在异质性以及瓶颈效应中的遗传漂变问题。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。与传统筛选体系的基准对标验证表明，该技术可显著提升实验数据质量。我们预计CRISPR-StAR将为复杂模型（尤其是体内模型）中的遗传筛选建立新的标准。整体实验设计：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筛选以及小型验证筛选实验。我们对从筛选后的体内肿瘤及体外培养细胞中获取的样本进行了二代测序（NGS, Next Generation Sequencing）处理，并先后采用全基因组CRISPR-Cas9敲除模型分析工具（MAGeCK, Model-based Analysis of Genome-wide CRISPR-Cas9 Knockout）进行分析；不过本次分析实际依据CRISPR-StAR专属分析流程完成。全基因组筛选实验分两批次开展。