1. Chromosomes and Gene Expression
Download icon

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 Tjian
  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
Short Report
  • Cited 164
  • Views 12,352
  • Annotations
Cite this article as: eLife 2016;5:e12677 doi: 10.7554/eLife.12677

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 Tjian

    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 Tjian, 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

Publication 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

  • 12,352
    Page views
  • 4,094
    Downloads
  • 164
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, Scopus, PubMed Central.

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)

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

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

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 et al.
    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 et al.
    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. Cell Biology
    2. Chromosomes and Gene Expression

    Repressing Ago2 mRNA translation by Trim71 maintains pluripotency through inhibiting let-7 microRNAs

    Qiuying Liu et al.
    Research Article Updated

    The regulation of stem cell fate is poorly understood. Genetic studies in Caenorhabditis elegans lead to the hypothesis that a conserved cytoplasmic double-negative feedback loop consisting of the RNA-binding protein Trim71 and the let-7 microRNA controls the pluripotency and differentiation of stem cells. Although let-7-microRNA-mediated inhibition of Trim71 promotes differentiation, whether and how Trim71 regulates pluripotency and inhibits the let-7 microRNA are still unknown. Here, we show that Trim71 represses Ago2 mRNA translation in mouse embryonic stem cells. Blocking this repression leads to a specific post-transcriptional increase of mature let-7 microRNAs, resulting in let-7-dependent stemness defects and accelerated differentiation in the stem cells. These results not only support the Trim71-let-7-microRNA bi-stable switch model in controlling stem cell fate, but also reveal that repressing the conserved pro-differentiation let-7 microRNAs at the mature microRNA level by Ago2 availability is critical to maintaining pluripotency.