title: The Impact of Chromatin Dynamics on Cas9-Mediated Genome Editing in Human Cells.
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.
authors: School of Biological and Health Systems Engineering, Arizona State University , 501 E. Tyler Mall, ECG 344A, Tempe, Arizona 85287, United States.
authors: René M
authors: RM
authors: Daer
authors: School of Biological and Health Systems Engineering, Arizona State University , 501 E. Tyler Mall, ECG 344A, Tempe, Arizona 85287, United States.
authors: Josh P
authors: JP
authors: Cutts
authors: School of Biological and Health Systems Engineering, Arizona State University , 501 E. Tyler Mall, ECG 344A, Tempe, Arizona 85287, United States.
authors: David A
authors: DA
authors: Brafman
authors: School of Biological and Health Systems Engineering, Arizona State University , 501 E. Tyler Mall, ECG 344A, Tempe, Arizona 85287, United States.
authors: Karmella A
authors: KA
authors: Haynes
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems / Q000235:genetics*; D002460:Cell Line; D002843:Chromatin / Q000235:genetics*; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D004247:DNA / Q000235:genetics; D004720:Endonucleases / Q000235:genetics; D000072669:Gene Editing / Q000379:methods; D016678:Genome / Q000235:genetics*; D023281:Genomics / Q000379:methods; D057809:HEK293 Cells; D006801:Humans
chemical_list: D002843:Chromatin; D004247:DNA; D004720:Endonucleases
keywords: Cas9-mediated editing; INDEL; chromatin