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
Reviewing Editor
- Karen Adelman, National Institute of Environmental Health Sciences, United States
Publication history
- Received: October 29, 2015
- Accepted: March 16, 2016
- Accepted Manuscript published: March 17, 2016 (version 1)
- Accepted Manuscript updated: March 18, 2016 (version 2)
- Accepted Manuscript updated: March 31, 2016 (version 3)
- 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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