Effective interpretation of genome function and genetic variation requires a shift from epigenetic mapping of cis-regulatory elements (CREs) to characterization of endogenous function. We developed HCR-FlowFISH, a broadly applicable approach to characterize CRISPR-perturbed CREs via accurate quantification of native transcripts, alongside CASA (CRISPR Activity Screen Analysis), a hierarchical Bayesian model to quantify CRE activity. Across >325,000 perturbations, we provide evidence that CREs can regulate multiple genes, skip over the nearest gene, and can display activating and/or silencing effects. At the cholesterol-level associated FADS locus, we combine endogenous screens with reporter assays to exhaustively characterize multiple genome-wide association signals, functionally nominating causal variants and importantly, identifying their target genes.

CRISPR screens for cis-regulatory elements (CREs) have shown unprecedented power to endogenously characterize the non-coding genome. To characterize CREs we developed HCR-FlowFISH (Hybridization Chain Reaction Fluorescent In-Situ Hybridization coupled with Flow Cytometry), which directly quantifies native transcripts within their endogenous loci following CRISPR perturbations of regulatory elements, eliminating the need for restrictive phenotypic assays such as growth or transcript-tagging. HCR-FlowFISH accurately quantifies gene expression across a wide range of transcript levels and cell types. We also developed CASA (CRISPR Activity Screen Analysis), a hierarchical Bayesian model to identify and quantify CRE activity. Using >270,000 perturbations, we identified CREs for GATA1, HDAC6, ERP29, LMO2, MEF2C, CD164, NMU, FEN1 and the FADS gene cluster. Our methods detect subtle gene expression changes and identify CREs regulating multiple genes, sometimes at different magnitudes and directions. We demonstrate the power of HCR-FlowFISH to parse genome-wide association signals by nominating causal variants and target genes.

In the version of this Article initially published, there was an error in the SNP number shown. In Figure 3b, the center label "rs1546273" should have read "rs1546723, " and in the third paragraph of the section titled "Comprehensive CRE scans of five loci show the flexibility of HCR-FlowFISH, " in the sentence beginning "At the −77 kb CRE, " the identifier "rs1546273" should have read "rs1546723. " The errors have been corrected in the online version of the paper.