Current pooled CRISPR screens for cis-regulatory elements (CREs), based on transcriptional output changes, are typically limited to characterizing CREs of only one gene. Here, we describe CRISPRpath, a scalable screening strategy for parallelly characterizing CREs of genes linked to the same biological pathway and converging phenotypes. We demonstrate the ability of CRISPRpath for simultaneously identifying functional enhancers of six genes in the 6-thioguanine–induced DNA mismatch repair pathway using both CRISPR interference (CRISPRi) and CRISPR nuclease (CRISPRn) approaches. Sixty percent of the identified enhancers are known promoters with distinct epigenomic features compared to other active promoters, including increased chromatin accessibility and interactivity. Furthermore, by imposing different levels of selection pressure, CRISPRpath can distinguish enhancers exerting strong impact on gene expression from those exerting weak impact. Our results offer a nuanced view of cis-regulation and demonstrate that CRISPRpath can be leveraged for understanding the complex gene regulatory program beyond transcriptional output at scale.

中文翻译：

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使用 CRISPRpath 并行表征多个基因的顺式调控元件


目前基于转录输出变化的顺式调控元件 (CRE) 的 CRISPR 混合筛选通常仅限于表征仅一个基因的 CRE。在这里，我们描述了 CRISPRpath，这是一种可扩展的筛选策略，用于并行表征与相同生物途径和趋同表型相关的基因的 CRE。我们证明了 CRISPRpath 使用 CRISPR 干扰 (CRISPRi) 和 CRISPR 核酸酶 (CRISPRn) 方法同时识别 6-硫鸟嘌呤诱导的 DNA 错配修复途径中六个基因的功能增强子的能力。 60% 的已鉴定增强子是已知的启动子，与其他活性启动子相比具有独特的表观基因组特征，包括增加的染色质可及性和交互性。此外，通过施加不同水平的选择压力，CRISPRpath可以区分对基因表达产生强烈影响的增强子和产生弱影响的增强子。我们的结果提供了顺式调控的细致入微的观点，并证明 CRISPRpath 可用于理解除大规模转录输出之外的复杂基因调控程序。