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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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

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Research organism

Further reading

    1. Computational and Systems Biology
    2. Chromosomes and Gene Expression

    Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation

    Max A Horlbeck, Luke A Gilbert ... Jonathan S Weissman
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    We recently found that nucleosomes directly block access of CRISPR/Cas9 to DNA (Horlbeck et al., 2016). Here, we build on this observation with a comprehensive algorithm that incorporates chromatin, position, and sequence features to accurately predict highly effective single guide RNAs (sgRNAs) for targeting nuclease-dead Cas9-mediated transcriptional repression (CRISPRi) and activation (CRISPRa). We use this algorithm to design next-generation genome-scale CRISPRi and CRISPRa libraries targeting human and mouse genomes. A CRISPRi screen for essential genes in K562 cells demonstrates that the large majority of sgRNAs are highly active. We also find CRISPRi does not exhibit any detectable non-specific toxicity recently observed with CRISPR nuclease approaches. Precision-recall analysis shows that we detect over 90% of essential genes with minimal false positives using a compact 5 sgRNA/gene library. Our results establish CRISPRi and CRISPRa as premier tools for loss- or gain-of-function studies and provide a general strategy for identifying Cas9 target sites.

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    Nucleosome breathing and remodeling constrain CRISPR-Cas9 function

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    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.

    1. Chromosomes and Gene Expression

    Single molecule analysis of CENP-A chromatin by high-speed atomic force microscopy

    Daniël P Melters, Keir C Neuman ... Yamini Dalal
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    Chromatin accessibility is modulated in a variety of ways to create open and closed chromatin states, both of which are critical for eukaryotic gene regulation. At the single molecule level, how accessibility is regulated of the chromatin fiber composed of canonical or variant nucleosomes is a fundamental question in the field. Here, we developed a single-molecule tracking method where we could analyze thousands of canonical H3 and centromeric variant nucleosomes imaged by high-speed atomic force microscopy. This approach allowed us to investigate how changes in nucleosome dynamics in vitro inform us about transcriptional potential in vivo. By high-speed atomic force microscopy, we tracked chromatin dynamics in real time and determined the mean square displacement and diffusion constant for the variant centromeric CENP-A nucleosome. Furthermore, we found that an essential kinetochore protein CENP-C reduces the diffusion constant and mobility of centromeric nucleosomes along the chromatin fiber. We subsequently interrogated how CENP-C modulates CENP-A chromatin dynamics in vivo. Overexpressing CENP-C resulted in reduced centromeric transcription and impaired loading of new CENP-A molecules. From these data, we speculate that factors altering nucleosome mobility in vitro, also correspondingly alter transcription in vivo. Subsequently, we propose a model in which variant nucleosomes encode their own diffusion kinetics and mobility, and where binding partners can suppress or enhance nucleosome mobility.