1. Computational and Systems Biology
  2. Chromosomes and Gene Expression
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Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation

  1. Max A Horlbeck
  2. Luke A Gilbert
  3. Jacqueline E Villalta
  4. Britt Adamson
  5. Ryan A Pak
  6. Yuwen Chen
  7. Alexander P Fields
  8. Chong Y Park
  9. Jacob E Corn
  10. Martin Kampmann
  11. Jonathan S Weissman  Is a corresponding author
  1. University of California, San Francisco, United States
  2. University of California, Berkeley, United States
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Cite this article as: eLife 2016;5:e19760 doi: 10.7554/eLife.19760

Abstract

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.

Article and author information

Author details

  1. Max A Horlbeck

    Department of Cellular and Molecular Pharmacology, California Institute for Quantitative Biomedical Research, University of California, San Francisco, San Francisco, United States
    Competing interests
    Max A Horlbeck, MAH, LAG, MK, and JSW have filed a patent application related to CRISPRi and CRISPRa screening (PCT/US15/40449). JSW is a founder of, and MAH and LAG are consultants for, KSQ Therapeutics, a CRISPR functional genomics company..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3875-871X
  2. Luke A Gilbert

    Department of Cellular and Molecular Pharmacology, California Institute for Quantitative Biomedical Research, University of California, San Francisco, San Francisco, United States
    Competing interests
    Luke A Gilbert, MAH, LAG, MK, and JSW have filed a patent application related to CRISPRi and CRISPRa screening (PCT/US15/40449). JSW is a founder of, and MAH and LAG are consultants for, KSQ Therapeutics, a CRISPR functional genomics company..
  3. Jacqueline E Villalta

    Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States
    Competing interests
    No competing interests declared.
  4. Britt Adamson

    Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States
    Competing interests
    No competing interests declared.
  5. Ryan A Pak

    Departmant of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3507-3122
  6. Yuwen Chen

    Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States
    Competing interests
    No competing interests declared.
  7. Alexander P Fields

    Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States
    Competing interests
    No competing interests declared.
  8. Chong Y Park

    Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States
    Competing interests
    No competing interests declared.
  9. Jacob E Corn

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7798-5309
  10. Martin Kampmann

    Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States
    Competing interests
    Martin Kampmann, MAH, LAG, MK, and JSW have filed a patent application related to CRISPRi and CRISPRa screening (PCT/US15/40449)..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3819-7019
  11. Jonathan S Weissman

    Center for RNA Systems Biology, University of California, San Francisco, San Francisco, United States
    For correspondence
    Jonathan.Weissman@ucsf.edu
    Competing interests
    Jonathan S Weissman, MAH, LAG, MK, and JSW have filed a patent application related to CRISPRi and CRISPRa screening (PCT/US15/40449). JSW is a founder of, and MAH and LAG are consultants for, KSQ Therapeutics, a CRISPR functional genomics company..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2445-670X

Funding

Howard Hughes Medical Institute

  • Max A Horlbeck
  • Luke A Gilbert
  • Jacqueline E Villalta
  • Britt Adamson
  • Yuwen Chen
  • Alexander P Fields

National Institutes of Health (P50 GM102706, U01 CA168370, R01 DA036858)

  • Max A Horlbeck
  • Luke A Gilbert
  • Jacqueline E Villalta
  • Britt Adamson
  • Yuwen Chen
  • Alexander P Fields

Leukemia and Lymphoma Society

  • Luke A Gilbert

Li Ka Shing Foundation

  • Ryan A Pak
  • Chong Y Park
  • Jacob E Corn

National Institutes of Health (NIGMS DP2 GM119139)

  • Martin Kampmann

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Karen Adelman, Harvard Medical School, United States

Publication history

  1. Received: July 19, 2016
  2. Accepted: September 22, 2016
  3. Accepted Manuscript published: September 23, 2016 (version 1)
  4. Version of Record published: November 3, 2016 (version 2)

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. Further reading

Further reading

    1. Chromosomes and Gene Expression
    Max A Horlbeck et al.
    Short Report Updated

    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.

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    Research Article Updated