Highly specific epigenome editing by CRISPR-Cas9 repressors for silencing of distal regulatory elements
Open accessAbstract
Epigenome editing with the CRISPR (clustered, regularly interspaced, short palindromic repeats)-Cas9 platform is a promising technology for modulating gene expression to direct cell phenotype and to dissect the causal epigenetic mechanisms of gene regulation. Fusions of nuclease-inactive dCas9 to the Krüppel-associated box (KRAB) repressor (dCas9-KRAB) can silence target gene expression, but the genome-wide specificity and the extent of heterochromatin formation catalyzed by dCas9-KRAB are not known. We targeted dCas9-KRAB to the HS2 enhancer, a distal regulatory element that orchestrates the expression of multiple globin genes, and observed highly specific induction of H3K9 trimethylation (H3K9me3) at the enhancer and decreased chromatin accessibility of both the enhancer and its promoter targets. Targeted epigenetic modification of HS2 silenced the expression of multiple globin genes, with minimal off-target changes in global gene expression. These results demonstrate that repression mediated by dCas9-KRAB is sufficiently specific to disrupt the activity of individual enhancers via local modification of the epigenome.
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dCas9-KRAB (CRISPRi) was the first construct shown to synthetically silence a target enhancer by inducing ~1-2 kb of repressive marks near the gRNA target.
“The dCas9–KRAB repressor domain (CRISPR interference or ‘CRISPRi’) was the first construct shown to synthetically silence a target enhancer by inducing ~1-2 kb of repressive marks in the vicinity of the gRNA target207 (Figure 2c).”
From Towards a comprehensive catalogue of validated and target-linked human enhancers (2020) · cites this paper as [207]
The Results section ('dCas9-KRAB induces histone methylation at enhancers'; page unavailable) reports repressive H3K9me3 at the targeted HS2 enhancer, covering 853 bp there. The Discussion reports increases up to 4.5 kb from the sgRNA target, not a general ~1–2 kb span. It also notes earlier enhancer repression with KRAB fusion proteins, so it does not establish the 'first construct' claim. (LLM, conf=0.88)
Experimental — accuracy not yet measuredEpigenetic editing therapies are currently being evaluated in preclinical models of PTSD, addiction, and MDD.
“They are currently being evaluated in preclinical models of PTSD, addiction, and MDD (125, 126).”
From The clinical use of epigenetics in psychiatry: a narrative review of epigenetic mechanisms, key candidate genes, and precision psychiatry (2025) · cites this paper as [126]
The Results section describes dCas9-KRAB experiments on globin genes in K562 cells, not evaluations of therapies in preclinical psychiatric models. Searches for PTSD, addiction, MDD, depression, and related preclinical models found no passage addressing the claim. (LLM, conf=0.78)
Experimental — accuracy not yet measureddCas9-KRAB system reversibly suppresses gene expression through heterochromatin formation and significantly reduced expression of a series of endogenous genes.
“dCas9-KRAB system, recruiting a diverse array of histone modifiers that reversibly suppresses gene expression through the formation of heterochromatin, significantly reduced the expression of a series of endogenous genes in the genome [125, 128].”
From Non-viral delivery systems for CRISPR/Cas9-based genome editing: challenges and opportunities (2018) · cites this paper as [125]
dCas9 fusion proteins-mediated CRISPRi, such as dCas9-KRAB and dCas9-LSD1, has been introduced.
“Most recently, dCas9 fusion proteins-mediated CRISPRi, such as dCas9-KRAB (Krüppel-associated box) and dCas9-LSD1 (lysine-specific histone demethylase 1) system, has been introduced [125–127].”
From Non-viral delivery systems for CRISPR/Cas9-based genome editing: challenges and opportunities (2018) · cites this paper as [125]
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- Authors
- Thakore, P. I., D'Ippolito, A. M., Song, L., Safi, A., Shivakumar, N. K., Kabadi, A. M., Reddy, T. E., Crawford, G. E., & Gersbach, C. A.
- Affiliations
- Duke University (US)
- Corresponding author
- Reddy, Timothy E
- Crawford, Gregory E
- Gersbach, Charles A
- Venue
- Nature Methods
- Published
- 2015-10-26
- Volume
- 12
- Issue
- 12
- Pages
- 1143-1149
- ISSN
- 1548-7091 (ISSN-L)
- 1548-7105
- PMC
- PMC4666778
- OpenAlex
- W2239511084
- Cited by
- 1,012
- References
- 55
- Citations by year
- 2026: 68 · 2025: 80 · 2024: 80 · 2023: 100 · 2022: 111 · 2021: 121 · 2020: 107 · 2019: 107 · 2018: 100 · 2017: 72 · 2016: 59 · 2015: 3 · 2013: 1
- FWCI
- 33.89
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature Methods (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- 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 (T32GM007754)
- National Institute of Arthritis and Musculoskeletal and Skin Diseases (R21AR065956, P30AR066527)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Epigenetics and DNA Methylation
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
- Keywords
- epigenome editing, CRISPR/Cas9, distal regulatory elements, enhancer silencing, globin genes, H3K9 trimethylation, heterochromatin formation, chromatin accessibility, off-target specificity, gene expression inhibition
- MeSH terms
- RNA, Guide, CRISPR-Cas Systems, Enhancer Elements, Genetic, Globins, Humans, Repressor Proteins, Viral Proteins, Gene Expression Regulation, Viral, Lentivirus, RNA, Guide, Kinetoplastida, K562 Cells, Epigenesis, Genetic, Regulatory Elements, Transcriptional, HEK293 Cells, Epigenomics, Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR-Cas Systems, CRISPR-Associated Proteins
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