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Parallel Characterization of cis-Regulatory Elements for Multiple Genes Using CRISPRpath

By Xingjie Ren, Mengchi Wang, Bingkun Li, Kirsty Jamieson, Lina Zheng, Ian R. Jones, Bin Li, Maya Asami Takagi, Jerry Lee, Lenka Maliskova, Tsz Wai Tam, Miao Yu, Rong Hu, Lindsay Lee, Armen Abnousi, Gang Li, Yun Li, Ming Hu, Bing Ren, Wei Wang, Yin Shen

Posted 19 Feb 2021
bioRxiv DOI: 10.1101/2021.02.19.431931

Current pooled CRISPR screens for cis-regulatory elements (CREs) can only accommodate one gene based on its expression level. 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. 60% 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.

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