1. Chromosomes and Gene Expression

Nucleosomes impede Cas9 access to DNA in vivo and in vitro

  1. Max A Horlbeck
  2. Lea B Witkowsky
  3. Benjamin Guglielmi
  4. Joseph M Replogle
  5. Luke A Gilbert
  6. Jacqueline E Villalta
  7. Sharon E Torigoe
  8. Robert Tijan
  9. Jonathan S Weissman  Is a corresponding author
  1. Howard Hughes Medical Institute, University of California, San Francisco, United States
  2. Howard Hughes Medical Institute, University of California, Berkeley, United States
https://doi.org/10.7554/eLife.12677
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Cite this article
  1. Max A Horlbeck
  2. Lea B Witkowsky
  3. Benjamin Guglielmi
  4. Joseph M Replogle
  5. Luke A Gilbert
  6. Jacqueline E Villalta
  7. Sharon E Torigoe
  8. Robert Tijan
  9. Jonathan S Weissman
(2016)
Nucleosomes impede Cas9 access to DNA in vivo and in vitro
eLife 5:e12677.
https://doi.org/10.7554/eLife.12677
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  3. Download .RIS

Abstract

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.

Article and author information

Author details

  1. Max A Horlbeck

    Department of Cellular and Molecular Pharmacology, California Institute for Quantitative Biomedical Research, Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, United States
    Competing interests
    Max A Horlbeck, Filed a patent application related to CRISPRi screening techonology.

  2. Lea B Witkowsky

    Department of Molecular and Cell Biology, Li Ka Shing Center for Biomedical and Health Sciences, Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.

  3. Benjamin Guglielmi

    Department of Molecular and Cell Biology, Li Ka Shing Center for Biomedical and Health Sciences, Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.

  4. Joseph M Replogle

    Department of Cellular and Molecular Pharmacology, California Institute for Quantitative Biomedical Research, Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, United States
    Competing interests
    No competing interests declared.

  5. Luke A Gilbert

    Department of Cellular and Molecular Pharmacology, California Institute for Quantitative Biomedical Research, Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, United States
    Competing interests
    Luke A Gilbert, Filed a patent application related to CRISPRi screening techonology.

  6. Jacqueline E Villalta

    Department of Cellular and Molecular Pharmacology, California Institute for Quantitative Biomedical Research, Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, United States
    Competing interests
    No competing interests declared.

  7. Sharon E Torigoe

    Department of Molecular and Cell Biology, Li Ka Shing Center for Biomedical and Health Sciences, Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.

  8. Robert Tijan

    Department of Molecular and Cell Biology, Li Ka Shing Center for Biomedical and Health Sciences, Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, United States
    Competing interests
    Robert Tijan, President of the Howard Hughes Medical Institute (2009-present), one of the three founding funders of eLife, and a member of eLife's Board of Directors.

  9. Jonathan S Weissman

    Department of Cellular and Molecular Pharmacology, California Institute for Quantitative Biomedical Research, Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, United States
    For correspondence
    Jonathan.Weissman@ucsf.edu
    Competing interests
    Jonathan S Weissman, Filed a patent application related to CRISPRi screeningtechonology.

Reviewing Editor

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

Version history

  1. Received: October 29, 2015
  2. Accepted: March 16, 2016
  3. Accepted Manuscript published: March 17, 2016 (version 1)
  4. Accepted Manuscript updated: March 18, 2016 (version 2)
  5. Accepted Manuscript updated: March 31, 2016 (version 3)
  6. Version of Record published: May 9, 2016 (version 4)

Copyright

© 2016, Horlbeck 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. Max A Horlbeck
  2. Lea B Witkowsky
  3. Benjamin Guglielmi
  4. Joseph M Replogle
  5. Luke A Gilbert
  6. Jacqueline E Villalta
  7. Sharon E Torigoe
  8. Robert Tijan
  9. Jonathan S Weissman
(2016)
Nucleosomes impede Cas9 access to DNA in vivo and in vitro
eLife 5:e12677.
https://doi.org/10.7554/eLife.12677

Share this article

https://doi.org/10.7554/eLife.12677

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