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CRISPR–Cas9 epigenome editing enables high-throughput screening for functional regulatory elements in the human genome
Open accessAbstract
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 dCas9 KRAB repressor and dCas9 p300 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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“Activating domains (dCas9–VPR or dCas9–p300) have also been used to scan for poised enhancers in an approach termed CRISPR activation or ‘CRISPRa’92,208(Figure 2d).”
From Towards a comprehensive catalogue of validated and target-linked human enhancers (2020) · cites this paper as [208]
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“When comparing Borzoi, Enformer and a distance-to-TSS baseline on their ability to classify measured positive from negative enhancer–gene interactions in data from previous works60–65, we find that Borzoi has superior average precision (AUPRC) and area under the receiver operating characteristic curve (AUROC) at all distances (Fig. 4c and Extended Data Fig. 5a).”
From Predicting RNA-seq coverage from DNA sequence as a unifying model of gene regulation (2025) · cites this paper as [62]
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“CRISPRi has subsequently been used in multiple single-gene screens of regulatory elements105,208,209.”
From Towards a comprehensive catalogue of validated and target-linked human enhancers (2020) · cites this paper as [208]
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“we validated 4 out of 5 enhancers identified in a CRISPRi screen for HBE1 enhancers using a fluorescent reporter22”
From Targeted Perturb-seq enables genome-scale genetic screens in single cells (2020) · cites this paper as [22]
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“15 gRNAs targeting the HBE1 TSS, and HS1–4 of the Globin LCR were chosen as validated from Klann et al. (2017) and Xie et al. (2017).”
From A Genome-wide Framework for Mapping Gene Regulation via Cellular Genetic Screens (2019) · cites this paper as Klann et al. (2017)
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Metadata
- Authors
- Klann, T. S., Black, J. B., Chellappan, M., Safi, A., Song, L., Hilton, I. B., Crawford, G. E., Reddy, T. E., & Gersbach, C. A.
- Affiliations
- Duke University (US)
- Corresponding author
- Crawford, Gregory E
- Reddy, Timothy E
- Gersbach, Charles A
- ORCID
- Venue
- Nature Biotechnology
- Published
- 2017-04-03
- Volume
- 35
- Issue
- 6
- Pages
- 561-568
- ISSN
- 0733-222X (ISSN-L)
- 1087-0156
- 1546-1696
- PMC
- PMC5462860
- OpenAlex
- W2604130415
- Cited by
- 447
- References
- 56
- Citations by year
- 2026: 23 · 2025: 24 · 2024: 37 · 2023: 39 · 2022: 53 · 2021: 57 · 2020: 70 · 2019: 64 · 2018: 55 · 2017: 24
- FWCI
- 24.45
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature Biotechnology (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- Funders
- National Institutes of Health (1dp2od008586-01)
- National Institute on Drug Abuse (R01DA036865)
- National Human Genome Research Institute (U01HG007900)
- National Institute of General Medical Sciences (T32GM008555, R41GM119914)
- National Institute of Arthritis and Musculoskeletal and Skin Diseases (P30AR066527)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Chromatin Remodeling and Cancer
- Keywords
- regulatory elements, high-throughput screening, Ceres, DNase I hypersensitive sites, loss-of-function screen, gain-of-function screen, beta-globin locus, non-coding genome
- MeSH terms
- Gene Editing, Chromosome Mapping, Humans, Genome, Human, Sequence Analysis, DNA, Regulatory Elements, Transcriptional, Epigenomics, High-Throughput Nucleotide Sequencing, Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR-Cas Systems, CRISPR-Associated Proteins
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