The Impact of Chromatin Dynamics on Cas9-Mediated Genome Editing in Human Cells

René M. Daer, Josh P. Cutts, David Brafman, Karmella Haynes

Research output: Contribution to journalArticlepeer-review

47 Scopus citations

Abstract

In order to efficiently edit eukaryotic genomes, it is critical to test the impact of chromatin dynamics on CRISPR/Cas9 function and develop strategies to adapt the system to eukaryotic contexts. So far, research has extensively characterized the relationship between the CRISPR endonuclease Cas9 and the composition of the RNA-DNA duplex that mediates the system’s precision. Evidence suggests that chromatin modifications and DNA packaging can block eukaryotic genome editing by custom-built DNA endonucleases like Cas9; however, the underlying mechanism of Cas9 inhibition is unclear. Here, we demonstrate that closed, gene-silencing-associated chromatin is a mechanism for the interference of Cas9-mediated DNA editing. Our assays use a transgenic cell line with a drug-inducible switch to control chromatin states (open and closed) at a single genomic locus. We show that closed chromatin inhibits binding and editing at specific target sites and that artificial reversal of the silenced state restores editing efficiency. These results provide new insights to improve Cas9-mediated editing in human and other mammalian cells.

Original languageEnglish (US)
Pages (from-to)428-438
Number of pages11
JournalACS Synthetic Biology
Volume6
Issue number3
DOIs
StatePublished - Mar 17 2017

Keywords

  • Cas9-mediated editing
  • INDEL
  • chromatin

ASJC Scopus subject areas

  • Biomedical Engineering
  • Biochemistry, Genetics and Molecular Biology (miscellaneous)

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