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

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.

Article and author information

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.

Reviewing Editor

  1. Karen Adelman, National Institute of Environmental Health Sciences, United States

Publication history

  1. Received: December 2, 2015
  2. Accepted: April 16, 2016
  3. Accepted Manuscript published: April 28, 2016 (version 1)
  4. Version of Record published: May 25, 2016 (version 2)

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
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