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.
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All raw CRISPRi screening data, MPRA data and processed files have been uploaded to the ENCODE portal with accession no. ENCSR455UGU. Track hubs are available for each locus screened at the following links: https://genome.ucsc.edu/s/skr2/GATA_HCR;https://genome.ucsc.edu/s/skr2/CD164_HCR;https://genome.ucsc.edu/s/skr2/ERP29_HCR;https://genome.ucsc.edu/s/skr2/LMO2_HCR;https://genome.ucsc.edu/s/skr2/NMU_HCR;https://genome.ucsc.edu/s/skr2/MEF2C_HCR;https://genome.ucsc.edu/s/skr2/FADS_HCR; andhttps://genome.ucsc.edu/s/skr2/MYC_HCR. DNase hypersensitivity and histone modification data were collected from ENCODE (https://www.encodeproject.org). Topologically associated domains were collected from the TADKB (http://dna.cs.miami.edu/TADKB/). Genome-wide association study data were collected from the UKBB and the Global Lipids Genetics Consortium (https://biobank.ndph.ox.ac.uk/showcase/ and http://lipidgenetics.org, respectively). Fine-mapping data are available at the Finucane Lab (https://www.finucanelab.org/data).
The CASA software is available at https://github.com/sjgosai/CASA. The Python software is managed using Miniconda, which is available at https://repo.continuum.io/miniconda/. The Bowtie software is available at https://bioconda.github.io. The GuideScan software is available at https://bioconda.github.io. FlowJo is available at https://www.flowjo.com/solutions/flowjo/ (v.10.7 was used). CellProfiler is available at https://cellprofiler.org/releases. The SAIGE software is available at https://github.com/weizhouUMICH/SAIGE. The BOLT-LMM software is available at https://alkesgroup.broadinstitute.org/BOLT-LMM/BOLT-LMM_manual.html. The FINEMAP software is available at http://www.christianbenner.com (v.1.3.1 was used). The susieR software is available at https://github.com/stephenslab/susieR (v.0.8.1.0521 was used).
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We thank C. Fulco, A. Lin, C. Myhrvold, H. Metsky, B. Petros, J. Ray, S. Schaffner and J. Xue for editing and conversations about the manuscript. We thank C. Otis, N. Pirete and P. Rodgers at the Broad Flow Cytometry Core for cytometry and sorting assistance. We thank the Broad Imaging Platform for custom scripting and assistance in image analysis. We thank J. Ray and M. Bakalar in the Hacohen Lab for sorting and microscopy assistance. We thank C. Fulco, J. Engreitz and E. Lander for discussion on PrimeFlow and CRISPR screens. This work and S.K.R., S.J.G., A.G., A.M.-S., S.K., D.B. and R.T. were supported by the ENCODE Functional Characterization Center (grant no. UM1HG009435), a Broad SPARC grant and the Howard Hughes Medical Institute. S.K.R. is partially supported by grant nos. K99HG010669 and F32HG00922. R.T. is supported by grant nos. R00HG008179 and R01AI151051. S.J.G. was partially supported by grant no. 4T32GM007226-41.
P.C.S. is a cofounder of and consultant to Sherlock Biosciences and board member of the Danaher Corporation. The other authors declare no competing interests.
Peer review information Nature Genetics thanks Ran Elkon and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended Data Fig. 1 CRISPRi induction, sorting schema, and construction of CASA (CRISPR Activity Screen Analysis), a generative model of CRE activity.
a, Induction of CRISPRi linked to BFP via doxycycline shows robust activation. b, Example sorting strategy showing detection of a target transcript (GATA1) amplified with Alexa-647 conjugated hairpins, and a housekeeping transcript (TBP) amplified with Alexa-488 conjugated hairpins. The top and bottom 10% of 647:488 normalized ratio are differentially sorted. c, The generative process underlying CASA (CRISPR Activity Screen Analysis) described as a plate model, explicit statistical parameterization, and variable definitions. Shaded and unshaded circles indicate observed and latent variables, respectively. The variable W corresponds to the set of windows tested, while each Nw arises from the set of sgRNAs considered at the wth window.
Extended Data Fig. 2 HCR-FlowFISH screens display high similarity and increased sensitivity compared to growth screens at the GATA1 locus.
a, Overlap of the GATA1 guide library used in this study and Fulco et al.17 library. b, High correlation (Pearson r = 0.84, two-sided t-test P = 3.4 × 10−106) between individual guide scores for detected sgRNAs shared in the GATA1 HCR-FlowFISH screen and the Fulco et al.17 growth screen (black line is the ordinary least squares regression best fit, gray shaded band is 95% confidence interval). c, Guide-wise score comparison for all sgRNAs shared between growth and HCR-FlowFISH screens, showing read-depth of gRNA drives correlation more than off-target effects (cutting specificity). d, Individual gRNA guide scores plotted at the GATA1 promoter locus display the opposite direction CREs for GATA1 and HDAC6. e, Comparison of individual guide scores for guides shared between the HCR-FlowFISH and Fulco et al.17 growth screens. The distributions scores within CREs are more distinctly separated from those without when using HCR-FlowFISH. The minima, centers, and maxima of the boxes indicate the 25th, 50th, and 75th percentiles of the data distributions. Whiskers capture all remaining data, excluding outliers extending beyond 1.5 times the interquartile range below or above the 25th or 75th percentiles, respectively. n = 906 (grey boxes) and n = 313 (green boxes) shared guides analyzed outside and inside CRE boundaries, respectively.
Extended Data Fig. 3 HCR-FlowFISH and CASA enhance selectivity of CRISPRi screens at the GATA1 locus.
a, HCR-FlowFISH and PrimeFlow-CRISPRi individual guide score comparison for shared guides. Guides are grouped by overlap with CASA-nominated CREs. We find using HCR-FlowFISH improves separability between guide scores inside and outside of designated CREs compared to PrimeFlow. We also note guide score variability is reduced in HCR-FlowFISH. The minima, centers, and maxima of the boxes indicate the 25th, 50th, and 75th percentiles of the data distributions. Whiskers capture all remaining data, excluding outliers extending beyond 1.5 times the interquartile range below or above the 25th or 75th percentiles, respectively. n = 2,897 (grey boxes) and n = 88 (green boxes) shared guides analyzed outside and inside CRE boundaries, respectively. b, CASA CRE identification on simplified ABC data and comparison to HCR data. CASA only considers the highest and lowest expression bins from the first PCR replicate of each CRISPRi-FlowFISH screen replicate, yet distinguishes CREs from non-specific scores induced by perturbing the GATA1 gene body, in contrast to the original analysis.
a,b, Connectogram diagrams showing K562 DHS (light blue), K562 H3K27ac (dark blue), guide coverage (black), HCR-FlowFISH composite guide score tracks, and CASA CREs calls for MYC (teal), PVT1 (salmon), LMO2 (orange), CAPRIN1 (navy), and CAT (lilac). CASA-derived CRE activity scores are shown as lines connecting the CRE to the target gene, and colored by effect on transcript abundance (black decreases abundance, red increases abundance). In a, ‘Pro’ and ‘e1-4’ denote the promoter and enhancers identified at this locus in Fulco et al.17. In b, ‘P’, ‘I’, ‘D’, denote the proximal, intermediate and distal promoters of LMO2, respectively. c, Relative mRNA expression compared to unperturbed cells for CRISPRi perturbations of distal, intermediate + distal, and proximal + distal promoters. Three technical replicates shown, bars represent standard deviation.
a,b, Individual guide scores (points) and CASA CRE calls (bars) of HCR-FlowFISH screens for FADS1 (green), FADS2 (teal), and FADS3 (orange). K562 DHS (light blue) and H3K27ac (dark blue) peaks are also shown. Notably, these elements are shared between all three FADS genes. Surprisingly, perturbing the CRE in a results in a modest, but detectable, increase in FADS3 transcripts, in contrast to the decreases in FADS1 and FADS2 transcript abundance.
Extended Data Fig. 6 Functional characterization nominates rs174466 as a FADS3 CRE-activity altering SNP.
a, Genomic region surrounding the FADS3 promoter, highlighting tiling MPRA signal (red) and HCR-FlowFISH composite score for FADS3 (orange). rs174466 is denoted, along with all variants in linkage disequilibrium (r2 ≥ 0.2). Variants within an HCR-FlowFISH identified FADS3 CRE are labeled in orange, and variants displaying allelic skew from MPRA are denoted with a red outline. SP2 ChIP-seq signal overlapping rs174466 is included in grey. b, GWAS trait associations with rs174466 shows multiple overlaps with metabolic targets of FADS3. c, MPRA activity for reference and alternate version of the rs174466 shows increased CRE activity on the alternate allele. d, Motif for SP2 highlighting change to alternate allele better matches the canonical motif.
Supplementary Figs. 1–7
Supplementary Tables 1–3
Sequencing primers, qPCR primers, MPRA primers and guides.
CRISPRi HCR–FlowFISH screen results.
FADS locus SNPs, GWAS and fine-mapping results.
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Reilly, S.K., Gosai, S.J., Gutierrez, A. et al. Direct characterization of cis-regulatory elements and functional dissection of complex genetic associations using HCR–FlowFISH. Nat Genet 53, 1166–1176 (2021). https://doi.org/10.1038/s41588-021-00900-4