Journal Article

Multicenter integrated analysis of noncoding CRISPRi screens

Mackay-Smith, Ava, Tewhey, Ryan, Lataniotis, Lazaros, Schmidt, Henri, Ren, Xingjie, Gersbach, Charles A., Gosai, Sager J., White, Alexander D., Yao, David, Oh, Jin Woo, Barrera, Alejandro, Leslie, Christina, Tycko, Josh, Luo, Yunhai, Kundaje, Anshul, Siklenka, Keith, Shen, Yin, Engreitz, Jesse M., Doughty, Benjamin R., Guo, Katherine, Greenleaf, William J., Bounds, Lexi R., Andreeva, Kalina, Youngworth, Ingrid, Sabeti, Pardis C., Reddy, Timothy E., Pritykin, Yuri, Bassik, Michael C., Moore, Jill E., Gabdank, Idan, Beer, Michael A., Reilly, Steven K., Yardımcı, Galip Gürkan, Guerrero-Altamirano, Tania

2024

Overview

The ENCODE Consortium’s efforts to annotate noncoding cis -regulatory elements (CREs) have advanced our understanding of gene regulatory landscapes. Pooled, noncoding CRISPR screens offer a systematic approach to investigate cis -regulatory mechanisms. The ENCODE4 Functional Characterization Centers conducted 108 screens in human cell lines, comprising >540,000 perturbations across 24.85 megabases of the genome. Using 332 functionally confirmed CRE–gene links in K562 cells, we established guidelines for screening endogenous noncoding elements with CRISPR interference (CRISPRi), including accurate detection of CREs that exhibit variable, often low, transcriptional effects. Benchmarking five screen analysis tools, we find that CASA produces the most conservative CRE calls and is robust to artifacts of low-specificity single guide RNAs. We uncover a subtle DNA strand bias for CRISPRi in transcribed regions with implications for screen design and analysis. Together, we provide an accessible data resource, predesigned single guide RNAs for targeting 3,275,697 ENCODE SCREEN candidate CREs with CRISPRi and screening guidelines to accelerate functional characterization of the noncoding genome. This analysis provides 108 noncoding CRISPR screens collated by the ENCODE4 consortium and establishes experimental guidelines for future CRISPRi screens characterizing functional cis -regulatory elements.

• Publisher: Nature Publishing Group US,Nature Publishing Group
• Subject: 631/1647/2217 / 631/208/191/2018 / Analysis / Bioinformatics / Biological Microscopy / Biological Techniques / Biomedical and Life Sciences / Biomedical Engineering/Biotechnology / Cell lines / Clustered Regularly Interspaced Short Palindromic Repeats - genetics / Consortia / CRISPR / CRISPR-Cas Systems - genetics / Genome / Genomes / Guidelines / Humans / K562 Cells / Life Sciences / Proteomics / Regulatory mechanisms (biology) / Regulatory sequences / RNA, Guide, CRISPR-Cas Systems / Screening / Sieve analysis