1. Biochemistry and Chemical Biology
  2. Chromosomes and Gene Expression

Nucleosome breathing and remodeling constrain CRISPR‐Cas9 function

  1. R Stefan Isaac
  2. Fuguo Jiang
  3. Jennifer A Doudna
  4. Wendell A Lim
  5. Geeta J Narlikar  Is a corresponding author
  6. Ricardo AB Almeida
  1. University of California, San Francisco, United States
  2. University of California, Berkeley, United States
  3. Howard Hughes Medical Institute, University of California, Berkeley, United States
  4. Howard Hughes Medical Institute, United States
https://doi.org/10.7554/eLife.13450
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Cite this article
  1. R Stefan Isaac
  2. Fuguo Jiang
  3. Jennifer A Doudna
  4. Wendell A Lim
  5. Geeta J Narlikar
  6. Ricardo AB Almeida
(2016)
Nucleosome breathing and remodeling constrain CRISPR‐Cas9 function
eLife 5:e13450.
https://doi.org/10.7554/eLife.13450
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Abstract

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 allows Cas9 to effectively act on chromatin in vivo.

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Author details

  1. R Stefan Isaac

    Department of Biochemistry and Biophysics and Tetrad Graduate Program, University of California, San Francisco, San Francisco, United States
    Competing interests
    No competing interests declared.

  2. Fuguo Jiang

    Department of Molecular and Cell Biology, California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.

  3. Jennifer A Doudna

    Department of Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, United States
    Competing interests
    Jennifer A Doudna, Co‐founder of Caribou Biosciences; Editas Medicine; Intellia Therapeutics.

  4. Wendell A Lim

    Department of Cellular and Molecular Pharmacology, Howard Hughes Medical Institute, San Francisco, United States
    Competing interests
    Wendell A Lim, Founder of Cell Design Labs, and member of its scientific advisory board.

  5. Geeta J Narlikar

    Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States
    For correspondence
    Geeta.Narlikar@ucsf.edu
    Competing interests
    No competing interests declared.

  6. Ricardo AB Almeida

    Department of Cellular and Molecular Pharmacology, Center for Systems and Synthetic Biology, University of California, San Francisco, San Francisco, United States
    Competing interests
    No competing interests declared.

Copyright

© 2016, Isaac et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. R Stefan Isaac
  2. Fuguo Jiang
  3. Jennifer A Doudna
  4. Wendell A Lim
  5. Geeta J Narlikar
  6. Ricardo AB Almeida
(2016)
Nucleosome breathing and remodeling constrain CRISPR‐Cas9 function
eLife 5:e13450.
https://doi.org/10.7554/eLife.13450

Share this article

https://doi.org/10.7554/eLife.13450

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Further reading

    1. Chromosomes and Gene Expression

    Nucleosomes impede Cas9 access to DNA in vivo and in vitro

    Max A Horlbeck, Lea B Witkowsky ... Jonathan S Weissman
    Short Report Updated

    The prokaryotic CRISPR (clustered regularly interspaced palindromic repeats)-associated protein, Cas9, has been widely adopted as a tool for editing, imaging, and regulating eukaryotic genomes. However, our understanding of how to select single-guide RNAs (sgRNAs) that mediate efficient Cas9 activity is incomplete, as we lack insight into how chromatin impacts Cas9 targeting. To address this gap, we analyzed large-scale genetic screens performed in human cell lines using either nuclease-active or nuclease-dead Cas9 (dCas9). We observed that highly active sgRNAs for Cas9 and dCas9 were found almost exclusively in regions of low nucleosome occupancy. In vitro experiments demonstrated that nucleosomes in fact directly impede Cas9 binding and cleavage, while chromatin remodeling can restore Cas9 access. Our results reveal a critical role of eukaryotic chromatin in dictating the targeting specificity of this transplanted bacterial enzyme, and provide rules for selecting Cas9 target sites distinct from and complementary to those based on sequence properties.