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
Share this article
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
  2. Download BibTeX
  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.

Metrics

  • 15,108
    views
  • 4,424
    downloads
  • 315
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  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

Categories and tags

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
    Research Advance Updated

    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.

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

    Nucleosome breathing and remodeling constrain CRISPR-Cas9 function

    R Stefan Isaac, Fuguo Jiang ... Ricardo Almeida
    Short Report Updated

    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
    2. Immunology and Inflammation

    Foxp3 depends on Ikaros for control of regulatory T cell gene expression and function

    Rajan M Thomas, Matthew C Pahl ... Andrew D Wells
    Research Article

    Ikaros is a transcriptional factor required for conventional T cell development, differentiation, and anergy. While the related factors Helios and Eos have defined roles in regulatory T cells (Treg), a role for Ikaros has not been established. To determine the function of Ikaros in the Treg lineage, we generated mice with Treg-specific deletion of the Ikaros gene (Ikzf1). We find that Ikaros cooperates with Foxp3 to establish a major portion of the Treg epigenome and transcriptome. Ikaros-deficient Treg exhibit Th1-like gene expression with abnormal production of IL-2, IFNg, TNFa, and factors involved in Wnt and Notch signaling. While Ikzf1-Treg-cko mice do not develop spontaneous autoimmunity, Ikaros-deficient Treg are unable to control conventional T cell-mediated immune pathology in response to TCR and inflammatory stimuli in models of IBD and organ transplantation. These studies establish Ikaros as a core factor required in Treg for tolerance and the control of inflammatory immune responses.