Multi-center integrated analysis of non-coding CRISPR screens

Abstract

The ENCODE4 Consortiums 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 ENCODE4 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 cCREs, and screening guidelines to accelerate functional characterization of the non-coding genome.

Discovery of target genes and pathways at GWAS loci by pooled single-cell CRISPR screens

“…S6). We found minimal differences in target gene identification when looking at potential cis effects within a smaller (100 kb) or larger (1 Mb) window surrounding the targeted cCRE (table S3F) (28)(29)(30).…”

Gapped-kmer sequence modeling robustly identifies regulatory vocabularies and distal enhancers conserved between evolutionarily distant mammals

“…HE5 has weak GATA1 binding in erythroblast and low DNase I accessibility in embryonic liver. Further, inhibiting HE5 with CRISPRi has the weakest effect on downregulating HBE1 expression among the five putative human enhancers mapped from mouse 53,54 ( Fig 3H ; Methods ). It is likely that HE5 may have accumulated mutations leading to loss of regulatory activity.…”

“…TEs can contribute up to 30% of the strongest DHS peaks in some tissues, but we do not know what proportion of these TFBS-carrying TEs act as transcriptional enhancers to relevant genes, and which might be regulatory noise. Only a small proportion of TE-enhancers have so far been functionally tested 48 , but we expect to see more functional validation of these elements in near future due to advances in noncoding CRISPR-based screening methodologies 54,65 .…”

“…CRISPRi has the weakest effect on downregulating HBE1 expression among the five putative human enhancers mapped from mouse 53,54 gkm-align identifies additional novel conserved enhancers and robustly predicts functional conservation when combined with cell-specific information.…”

Multiplex, single-cell CRISPRa screening for cell type specific regulatory elements

“…Ultimately, a variety of factors including chromatin accessibility and epigenetic modifications, gRNA design quality, and target-specific nuances around CRISPRa-responsiveness, may play a role in determining the success of a CRISPRa perturbation in a given cellular context. Future scaling of this technology and its application to additional, clinically relevant cell types, will provide rich training sets that may enable derivation of rational CRISPRa gRNA design rules for distal, cell-type-specific gene activation, which, in contrast to promoters and CRISPRi 16, 28, 29 , are quite lacking at present. Further, these results illustrate the unique potential of noncoding CRISPRa screens to identify regulatory elements that can mediate upregulation of target genes, regardless of whether or not the gene is natively expressed in the cell type of interest or not.…”