Direct characterization of cis-regulatory elements and functional dissection of complex genetic associations using HCR-FlowFISH.

Steven K Reilly
Sager J Gosai
Alan Gutierrez
Ava Mackay-Smith
Jacob C Ulirsch
Masahiro Kanai
Kousuke Mouri
Daniel Berenzy
Susan Kales
Gina M Butler
Adrianne Gladden-Young
Redwan M Bhuiyan, The Jackson Laboratory
Michael L. Stitzel, The Jackson Laboratory
Hilary K Finucane
Pardis C Sabeti
Ryan Tewhey, The Jackson Laboratory


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 hybridization chain reaction fluorescence in situ hybridization coupled with flow cytometry (HCR-FlowFISH), a broadly applicable approach to characterize CRISPR-perturbed CREs via accurate quantification of native transcripts, alongside CRISPR activity screen analysis (CASA), 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 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 nominate causal variants and, importantly, identify their target genes.