Nucleosomes impede Cas9 access to DNA in vivo and in vitro.
Elife
; 52016 03 17.
Article
em En
| MEDLINE
| ID: mdl-26987018
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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Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Proteínas de Bactérias
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DNA
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Nucleossomos
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Endonucleases
Idioma:
En
Ano de publicação:
2016
Tipo de documento:
Article
País de afiliação:
Estados Unidos