Mapping and dissecting essential regulatory elements using multi-resolution CRISPR screens

Hundreds of thousands of candidate cis-regulatory elements (cCREs) have been identified across a myriad of cell types, but relatively few have been empirically characterized. Here, we present a scalable, phenotype-driven CRISPR screening method to test thousands of regulatory elements in parallel, by perturbing thousands of DNase hypersensitive sites (DHS) around hundreds of essential genes in K562. Integrating data from Cas9 exon-targeting with our CRISPRi screens, we nominate and validate promoters that regulate other nearby genes. Using higher-resolution CRISPR systems (dCas9 alone or Cas9) to tile across several essential enhancers, we resolve distinct peaks of activity and dissect core transcription factor binding sites (TFBSs) within enhancers, highlighting the advantages of complementary CRISPR systems to investigate CREs. Lastly, we use our tiling results to interrogate how TFBSs interact to drive enhancer function at the CBFA2T3 locus. This study demonstrates a scalable CRISPR screening framework for identifying and dissecting CREs that regulate cellular phenotypes. The following datasets are available: 1) Comparative gene expression profiling analysis of RNA-seq data for K562 cells expressing dCas9-KRAB and its knockdown derivatives (sgNFE2 enhancer and sgSafe negative controls). 2) 21 day K562 proliferation screens using multiple CRISPR effector systems (nuclease-active Cas9, dCas9 alone, and dCas9 fused to the KRAB domain from ZNF705), comprehensively targeting significant essential regulatory elements from a large-scale DHS screen with all possible GuideScan sgRNAs. The sgRNA plasmid library is also sequenced and provided.