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

• Published in: Nature genetics Vol. 53; no. 8; pp. 1166 - 1176
• Main Authors: Reilly, Steven K., Gosai, Sager J., Gutierrez, Alan, Mackay-Smith, Ava, Ulirsch, Jacob C., Kanai, Masahiro, Mouri, Kousuke, Berenzy, Daniel, Kales, Susan, Butler, Gina M., Gladden-Young, Adrianne, Bhuiyan, Redwan M., Stitzel, Michael L., Finucane, Hilary K., Sabeti, Pardis C., Tewhey, Ryan
• Format: Journal Article
• Language: English
• Published: New York Nature Publishing Group US 01.08.2021; Nature Publishing Group
• Subjects: 631/1647; 631/208; 631/208/191; Adaptor Proteins, Signal Transducing - genetics; Agriculture; Animal Genetics and Genomics; Bayes Theorem; Bayesian analysis; Biomedical and Life Sciences; Biomedicine; Cancer Research; Cholesterol; Clustered Regularly Interspaced Short Palindromic Repeats; CRISPR; Delta-5 Fatty Acid Desaturase; Deoxyribonuclease I - genetics; Deoxyribonuclease I - metabolism; Epigenetic inheritance; Epigenetics; Fatty Acid Desaturases - genetics; Flow Cytometry; Fluorescence; Fluorescence in situ hybridization; GATA1 Transcription Factor - genetics; Gene expression; Gene Function; Gene mapping; Genes; Genetic diversity; Genetic regulation; Genetic research; Genomes; Genomics; Human Genetics; Humans; Hybridization; In Situ Hybridization, Fluorescence - methods; K562 Cells; LIM Domain Proteins - genetics; Methods; Models, Genetic; Nominations; Perturbation; Polymorphism, Single Nucleotide; Proto-Oncogene Proteins - genetics; Quantitative Trait Loci; Regulatory sequences; Regulatory Sequences, Nucleic Acid; RNA, Guide, CRISPR-Cas Systems; Sieve analysis; Target recognition; United States
• ISSN: 1061-4036; 1546-1718; 1546-1718
• DOI: 10.1038/s41588-021-00900-4

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. HCR–FlowFISH is a new approach to characterize CRISPR-perturbed cis -regulatory elements (CREs) via accurate quantification of native transcripts, alongside CRISPR activity screen analysis (CASA), a hierarchical Bayesian model to quantify CRE activity.