Multi-center integrated analysis of non-coding CRISPR screens

The ENCODE Consortium’s efforts to annotate non-coding, cis -regulatory elements (CREs) have advanced our understanding of gene regulatory landscapes which play a major role in health and disease. Pooled, non-coding CRISPR screens are a promising approach for systematically investigating gene regulatory mechanisms. Here, the ENCODE Functional Characterization Centers report 109 screens comprising 346,970 individual perturbations across 13.3Mb of the genome, using a variety of methods, readouts, and statistical analyses. Across 332 functionally confirmed CRE-gene links, we identify principles for screening endogenous, non-coding elements for causal regulatory mechanisms. Nearly all CREs show strong evidence of open chromatin, and targeting accessibility peak summits is a critical component of our proposed sgRNA design rules. We provide experimental guidelines to accurately detect CREs with variable, often low, transcriptional effects. We discover a previously undescribed DNA strand-bias for CRISPRi in transcribed regions with implications for screen design and analysis. Benchmarking five screen analysis tools, we find CASA produces the most conservative CRE calls and is robust to artifacts of low-specificity sgRNAs. Together, we provide an accessible data resource, predesigned sgRNAs targeting 3,275,697 ENCODE SCREEN candidate CREs, and screening guidelines to accelerate functional characterization of the non-coding genome. ### Competing Interest Statement A.K. is scientific co-founder of Ravel Biotechnology, is on the scientific advisory board of PatchBio, SerImmune, AINovo, TensorBio and OpenTargets, is a consultant with Illumina and owns shares in DeepGenomics, Immuni and Freenome. C.A.G. is a co-founder of Tune Therapeutics and Locus Biosciences, and an advisor to Tune Therapeutics and Sarepta Therapeutics. C.A.G. is an inventor on patents and patent applications related to CRISPR epigenome editing. J.T. and M.C.B. acknowledge an outside interest in Stylus Medicine. L.L. is currently employed by Sana Biotechnology. P.C.S is a co-founder of and consultant to Sherlock Biosciences and Board Member of Danaher Corporation. She is a shareholder in both companies. W.J.G. is a co-founder of Epinomics and an adviser to 10X Genomics, Guardant Health and Centrillion.