Nucleosomes impede Cas9 access to DNA in vivo and in vitro
Max A Horlbeck,
Lea B Witkowsky,
Benjamin Guglielmi,
Joseph M Replogle,
Luke A Gilbert,
Jacqueline E Villalta,
Sharon E Torigoe,
Robert Tjian,
Jonathan S Weissman
Affiliations
Max A Horlbeck
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States; California Institute for Quantitative Biomedical Research, University of California, San Francisco, San Francisco, United States; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, United States; Center for RNA Systems Biology, University of California, San Francisco, San Francisco, United States
Lea B Witkowsky
Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, United States; Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States; CIRM Center of Excellence, University of California, Berkeley, Berkeley, United States
Benjamin Guglielmi
Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, United States; Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States; CIRM Center of Excellence, University of California, Berkeley, Berkeley, United States
Joseph M Replogle
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States; California Institute for Quantitative Biomedical Research, University of California, San Francisco, San Francisco, United States; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, United States; Center for RNA Systems Biology, University of California, San Francisco, San Francisco, United States
Luke A Gilbert
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States; California Institute for Quantitative Biomedical Research, University of California, San Francisco, San Francisco, United States; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, United States; Center for RNA Systems Biology, University of California, San Francisco, San Francisco, United States
Jacqueline E Villalta
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States; California Institute for Quantitative Biomedical Research, University of California, San Francisco, San Francisco, United States; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, United States; Center for RNA Systems Biology, University of California, San Francisco, San Francisco, United States
Sharon E Torigoe
Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, United States; Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States; CIRM Center of Excellence, University of California, Berkeley, Berkeley, United States
Robert Tjian
Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, United States; Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States; CIRM Center of Excellence, University of California, Berkeley, Berkeley, United States
Jonathan S Weissman
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States; California Institute for Quantitative Biomedical Research, University of California, San Francisco, San Francisco, United States; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, United States; Center for RNA Systems Biology, University of California, San Francisco, San Francisco, United States
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.
