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CRISPR–Cas9 epigenome editing enables high-throughput screening for functional regulatory elements in the human genome

Abstract

Large genome-mapping consortia and thousands of genome-wide association studies have identified non-protein-coding elements in the genome as having a central role in various biological processes. However, decoding the functions of the millions of putative regulatory elements discovered in these studies remains challenging. CRISPR–Cas9-based epigenome editing technologies have enabled precise perturbation of the activity of specific regulatory elements. Here we describe CRISPR–Cas9-based epigenomic regulatory element screening (CERES) for improved high-throughput screening of regulatory element activity in the native genomic context. Using dCas9KRAB repressor and dCas9p300 activator constructs and lentiviral single guide RNA libraries to target DNase I hypersensitive sites surrounding a gene of interest, we carried out both loss- and gain-of-function screens to identify regulatory elements for the β-globin and HER2 loci in human cells. CERES readily identified known and previously unidentified regulatory elements, some of which were dependent on cell type or direction of perturbation. This technology allows the high-throughput functional annotation of putative regulatory elements in their native chromosomal context.

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Figure 1: CRISPR–Cas9-based epigenetic regulatory element screening (CERES) identifies regulatory elements of the β-globin locus in a loss-of-function screen.
Figure 2: A dCas9KRAB loss-of-function screen in A431 cells identified regulatory elements of HER2.
Figure 3: A dCas9p300 gain-of-function screen in HEK293T cells identified regulatory elements of HER2.
Figure 4: dCas9p300 and dCas9KRAB remodel epigenetic marks near novel regulatory elements identified from screens.
Figure 5: Comparison of HER2-activation screens in different cell types.
Figure 6: Comparison of HER2 activation and repression screens.

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Acknowledgements

This work was supported by the Thorek Memorial Foundation, the US National Institutes of Health (NIH) (grants R01DA036865, R41GM119914, and U01HG007900 to G.E.C., T.E.R., and C.A.G.; core facility grant P30AR066527; Biotechnology Training Grant T32GM008555 to T.S.K. and J.B.B.; Director's New Innovator Award DP2OD008586 to C.A.G.), and the National Science Foundation (NSF) (Faculty Early Career Development (CAREER) Award CBET-1151035 to C.A.G.).

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T.S.K., G.E.C., T.E.R., and C.A.G. designed experiments. T.S.K., J.B.B., A.S., L.S., and M.C. performed the experiments. I.B.H. provided critical reagents. T.S.K., G.E.C., T.E.R., and C.A.G. analyzed the data. T.S.K. and C.A.G. wrote the manuscript, with contributions by all others authors.

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Correspondence to Gregory E Crawford or Timothy E Reddy or Charles A Gersbach.

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T.S.K., J.B.B., I.B.H., G.E.C., T.E.R., and C.A.G. are named inventors on patent applications related to genome engineering. T.S.K., G.E.C., T.E.R., and C.A.G. are founders of Element Genomics.

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Klann, T., Black, J., Chellappan, M. et al. CRISPR–Cas9 epigenome editing enables high-throughput screening for functional regulatory elements in the human genome. Nat Biotechnol 35, 561–568 (2017). https://doi.org/10.1038/nbt.3853

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