Manipulation of chromatin to enhance CRISPR activity

Recently, we demonstrated that closed, silenced chromatin impedes Cas9 editing by reducing the accessibility of DNA to Cas9/sgRNA binding at a chromosomal transgene. Here, we compared two methods for modifying chromatin to enhance Cas9 efficiency: a chromatin-disrupting inhibitor drug, UNC1999 and a transiently expressed targeted transcriptional activator, Gal4-p65. In both cases, we observed loss of the silencing mark histone 3 lysine 27 trimethylation (H3K27me3). Only Gal4-p65 treatment increased target gene expression. Our results confirm the previous observation that Gal4-p65-induced over-expression impedes Cas9 activity. We show that a recovery period of 9 days after Gal4-p65 expression generates a more Cas9-permissive state. Cas9 activity was restored following local modification of chromatin with transiently expressed Gal4-p65 while no enhancement was observed with UNC1999. These results implicate activator-mediated chromatin remodeling as an effective method to enhance Cas9 editing at closed chromatin.