RT Journal Article
SR Electronic
T1 The impact of chromatin dynamics on Cas9-mediated genome editing in human cells
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 071464
DO 10.1101/071464
A1 René M. Daer
A1 Josh P. Cutts
A1 David A. Brafman
A1 Karmella A. Haynes
YR 2016
UL http://biorxiv.org/content/early/2016/08/25/071464.abstract
AB 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 RNADNA 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 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. CRISPRClustered regularly-interspaced palindromic repeatsPRCPolycomb Repressive ComplexsgRNAshort guide RNAINDELinsertion and deletion