Nucleosome breathing and remodeling constrain CRISPR-Cas9 function
Open accessAbstract
The CRISPR-Cas9 bacterial surveillance system has become a versatile tool for genome editing and gene regulation in eukaryotic cells, yet how CRISPR-Cas9 contends with the barriers presented by eukaryotic chromatin is poorly understood. Here we investigate how the smallest unit of chromatin, a nucleosome, constrains the activity of the CRISPR-Cas9 system. We find that nucleosomes assembled on native DNA sequences are permissive to Cas9 action. However, the accessibility of nucleosomal DNA to Cas9 is variable over several orders of magnitude depending on dynamic properties of the DNA sequence and the distance of the PAM site from the nucleosome dyad. We further find that chromatin remodeling enzymes stimulate Cas9 activity on nucleosomal templates. Our findings imply that the spontaneous breathing of nucleosomal DNA together with the action of chromatin remodelers allow Cas9 to effectively act on chromatin in vivo.
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The most active CRISPRoff sgRNAs are located in nucleosome-depleted regions of gene promoters.
“Overlaying nucleosome occupancy with CRISPRoff sgRNA activity scores for all genes showed that the most active sgRNAs are located in nucleosome-depleted regions of gene promoters, as we and others have shown previously for Cas9 and dCas9-based tools (Figures 6J, 6K, S6D and S6E) (Horlbeck et al., 2016b; Isaac et al., 2016).”
From Genome-wide programmable transcriptional memory by CRISPR-based epigenome editing (2021) · cites this paper as Isaac et al., 2016
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Self-citationMultiplex Genome Engineering Using CRISPR/Cas Systems
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“…tool for genome engineering, gene regulation, and live imaging is a remarkable achievement with profound impact in a wide variety of research fields and applications ( Makarova et al., 2015 ; Doudna and Charpentier, 2014 ; Cong et al., 2013 ; Jinek et al., 2012 ; 2013 ; Mali et al., 2013 ).”
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“Large scale nucleosome positioning or DNA accessibility maps are now readily available and can inform CRISPR sgRNA design in order to avoid targeting regions of low accessibility ( Jiang and Pugh, 2009 ; Thurman et al., 2012 ; Wu et al., 2014 ; Hsieh et al., 2015 ).”
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- Authors
- Isaac, R. S., Jiang, F., Doudna, J. A., Lim, W. A., Narlikar, G. J., & Almeida, R.
- Affiliations
- University of California, San Francisco (US)
- QB3 (US)
- Corresponding author
- Lim, Wendell A
- Narlikar, Geeta J
- Venue
- eLife
- Publisher
- eLife Sciences Publications, Ltd
- Published
- 2016-04-28
- Volume
- 5
- ISSN
- 2050-084X (ISSN-L)
- PMC
- PMC4880442
- OpenAlex
- W2342818826
- Cited by
- 234
- References
- 6
- Citations by year
- 2026: 20 · 2025: 14 · 2024: 20 · 2023: 17 · 2022: 19 · 2021: 31 · 2020: 32 · 2019: 20 · 2018: 23 · 2017: 27 · 2016: 9 · 2014: 1 · 2013: 1
- FWCI
- 13.69
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- eLife (publishedVersion, cc-by)
- Europe PMC (PubMed Central) (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- eScholarship (California Digital Library) (submittedVersion)
- Funders
- Merck Fellow of the Damon Runyon Cancer Research Foundation (DRG-2201-14)
- National Science Foundation (1244557)
- National Institutes of Health (R01 DA036858, P50 GM081879, R01 GM073767)
- Howard Hughes Medical Institute
- 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 › DNA and Nucleic Acid Chemistry
- Keywords
- CRISPR/Cas9, chromatin remodeling, nucleosome, DNA accessibility, PAM site, nucleosome dyad, genome editing
- MeSH terms
- Gene Editing, CRISPR-Associated Protein 9, Animals, Bacterial Proteins, DNA, Endonucleases, Nucleosomes, Protein Binding, Recombination, Genetic, Xenopus, CRISPR-Cas Systems
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