Discovery of stimulation-responsive immune enhancers with CRISPR activation
Open accessAbstract
The majority of genetic variants associated with common human diseases map to enhancers, non-coding elements that shape cell-type-specific transcriptional programs and responses to extracellular cues. Systematic mapping of functional enhancers and their biological contexts is required to understand the mechanisms by which variation in non-coding genetic sequences contributes to disease. Functional enhancers can be mapped by genomic sequence disruption, but this approach is limited to the subset of enhancers that are necessary in the particular cellular context being studied. We hypothesized that recruitment of a strong transcriptional activator to an enhancer would be sufficient to drive target gene expression, even if that enhancer was not currently active in the assayed cells. Here we describe a discovery platform that can identify stimulus-responsive enhancers for a target gene independent of stimulus exposure. We used tiled CRISPR activation (CRISPRa) to synthetically recruit a transcriptional activator to sites across large genomic regions (more than 100 kilobases) surrounding two key autoimmunity risk loci, CD69 and IL2RA. We identified several CRISPRa-responsive elements with chromatin features of stimulus-responsive enhancers, including an IL2RA enhancer that harbours an autoimmunity risk variant. Using engineered mouse models, we found that sequence perturbation of the disease-associated Il2ra enhancer did not entirely block Il2ra expression, but rather delayed the timing of gene activation in response to specific extracellular signals. Enhancer deletion skewed polarization of naive T cells towards a pro-inflammatory T helper (T H 17) cell state and away from a regulatory T cell state. This integrated approach identifies functional enhancers and reveals how non-coding variation associated with human immune dysfunction alters context-specific gene programs.
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This is not an inexorable chain of events; for example, stimulus-responsive enhancers may exhibit open chromatin and 3D interactions with their promoters before activation.
“However, it should be emphasized that this is not an inexorable chain of events. For example, stimulus-responsive enhancers may exhibit open chromatin and 3D interactions with their promoters before activation92,93.”
From Towards a comprehensive catalogue of validated and target-linked human enhancers (2020) · cites this paper as [92]
The paper reports that stimulation-responsive enhancers can be identified in unstimulated cells and that IL2RA enhancer sites overlap accessible chromatin and elements that loop to the promoter (Abstract, p. unavailable; Extended Data Figure 5, p. unavailable). It does not establish that chromatin accessibility and promoter interactions were both present before activation in the same cells; that timing is the unsupported part. (LLM, conf=0.78)
Experimental — accuracy not yet measuredActivating domains dCas9-VPR or dCas9-p300 have been used to scan for poised enhancers in CRISPRa.
“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 [92]
The Abstract reports using CRISPRa to scan genomic regions and identify stimulation-responsive enhancers in unstimulated cells. But it specifies dCas9-VP64, not either dCas9-VPR or dCas9-p300; the Methods section on cell generation likewise identifies dCas9-VP64. No page numbers are available in our copy. (LLM, conf=0.88)
Experimental — accuracy not yet measuredTo date, all pooled CRISPR-based enhancer screens for in-genome perturbation use Cas9-induced perturbations, including active Cas9 for sequence disruption or nuclease dead-Cas9 tethered to an epigenetic repressor or activator domain.
“To date, all such screens use Cas9-induced perturbations, including active Cas9 for sequence disruption195, or nuclease dead-Cas9 (dCas9) tethered to an epigenetic repressor105 or activator domain92.”
From Towards a comprehensive catalogue of validated and target-linked human enhancers (2020) · cites this paper as [92]
Scalable approaches using genome editing tools, such as CRISPR/Cas9, enable cis-regulatory elements to be dissected in cell lines and even in primary cell contexts.
“Scalable approaches using genome editing tools, such as CRISPR/Cas9, enable cis-regulatory elements to be dissected in cell lines and even in primary cell contexts (Fulco et al., 2016; Simeonov et al., 2017).”
From Unraveling Hematopoiesis through the Lens of Genomics (2020) · cites this paper as Simeonov et al., 2017
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References 38 · Cited by 297
- Authors
- Simeonov, D. R., Gowen, B. G., Boontanrart, M., Roth, T. L., Gagnon, J. D., Mumbach, M. R., Satpathy, A. T., Lee, Y., Bray, N. L., Chan, A. Y., Lituiev, D. S., Nguyen, M. L., Gate, R. E., Subramaniam, M., Li, Z., Woo, J. M., Mitros, T., Ray, G. J., Curie, G. L., Naddaf, N., Chu, J. S., Ma, H., Boyer, E., Van Gool, F., Huang, H., Liu, R., Tobin, V. R., Schumann, K., Daly, M. J., Farh, K. K., Ansel, K. M., Ye, C. J., Greenleaf, W. J., Anderson, M. S., Bluestone, J. A., Chang, H. Y., Corn, J. E., & Marson, A.
- Affiliations
- University of California, San Francisco (US)
- Innovative Genomics Institute (US)
- Stanford Medicine (US)
- Broad Institute (US)
- Illumina (United States) (US)
- Chan Zuckerberg Biohub San Francisco (US)
- Corresponding author
- Corn, Jacob E.
- Marson, Alexander
- ORCID
- Simeonov, Dimitre R.
- Gowen, Benjamin G.
- Roth, Theodore L.
- Gagnon, John D.
- Mumbach, Maxwell R.
- Satpathy, Ansuman T.
- Lee, Youjin
- Bray, Nicolas L.
- Chan, Alice Y.
- Lituiev, Dmytro S.
- Nguyen, Michelle L.
- Subramaniam, Meena
- Li, Zhongmei
- Mitros, Therese
- Ray, Graham J.
- Naddaf, Nicki
- Chu, Julia S.
- Huang, Hailiang
- Tobin, Victoria R.
- Schumann, Kathrin
- Daly, Mark J.
- Farh, Kyle K.
- Ansel, K. Mark
- Ye, Chun J.
- Greenleaf, William J.
- Anderson, Mark S.
- Bluestone, Jeffrey A.
- Chang, Howard Y.
- Corn, Jacob E.
- Marson, Alexander
- Venue
- Nature
- Published
- 2017-08-30
- Volume
- 549
- Issue
- 7670
- Pages
- 111-115
- ISSN
- 0028-0836 (ISSN-L)
- 1476-4687
- PMC
- PMC5675716
- OpenAlex
- W2753608792
- Cited by
- 297
- References
- 38
- Citations by year
- 2026: 16 · 2025: 17 · 2024: 25 · 2023: 31 · 2022: 30 · 2021: 44 · 2020: 50 · 2019: 44 · 2018: 29 · 2017: 10
- FWCI
- 14.86
- Type
- journal article
- Language
- en
- Update notices
- correction (2018-06-13)
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Nature (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- RePEc: Research Papers in Economics (submittedVersion)
- eScholarship (California Digital Library) (submittedVersion)
- Funders
- National Institutes of Health (R01HG0081410-01)
- National Heart, Lung, and Blood Institute (R01HL109102)
- National Human Genome Research Institute (P50HG007735, R01HG008140, T32HG000044)
- National Institute of General Medical Sciences (T32GM007618, T32GM067547, T32GM007232)
- National Institute of Allergy and Infectious Diseases (T32AI007334)
- National Institute of Diabetes and Digestive and Kidney Diseases (K01DK114379, DP3DK111914, P30DK063720, P30DK043351, T32DK007418)
- National Center for Research Resources (S10RR029668, S10RR027303)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genetic Associations and Epidemiology
- Keywords
- CRISPR activation, CD69, IL2RA, non-coding variants, Th17 cells, regulatory T cells, genetically engineered mouse models, gene regulatory elements, transcriptional activator
- MeSH terms
- CD69 Antigens, Animals, Antigens, Differentiation, T-Lymphocyte, Cell Differentiation, Cell Line, Chromatin, Enhancer Elements, Genetic, Female, Gene Expression Regulation, Humans, Receptors, Antigen, T-Cell, Autoimmunity, Antigens, CD, Lectins, C-Type, Mice, Interleukin-2 Receptor alpha Subunit, Th17 Cells, Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR-Cas Systems
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