Enhancing Cas9 Activity in Heterochromatin

Recently, we demonstrated that closed, silenced chromatin impedes CRISPR/Cas9 editing by reducing the accessibility of DNA to Cas9/sgRNA binding at a chromosomal Tk-luciferase transgene. Our investigation of the underlying mechanism showed that disruption of chromatin through knock-down of the Polycomb complex enhanced Cas9-mediated editing but that over expression of the gene at the target site impeded Cas9 activity. Thus, further research is needed to establish chromatin manipulation as a practical tool for gene editing. Here, we compared two general methods for inducing an open, non-silenced state to enhance Cas9 e ciency: chromatin-disrupting inhibitor drugs and transient expression of site-speci c chromatin-modifying proteins. We explored the mechanism of enhanced editing at arti cially opened chromatin in HEK293 cells using deep sequencing of edited DNA and chromatin immunoprecipitation (ChIP) of histone modi cations. In both cases, we observed loss of the silencing mark histone 3 lysine 27 trimethylation (H3K27me3), while only treatment with the site-speci c, fusion transcriptional activator Gal4-p65 resulted in an increase in target gene expression. Our results rea rm 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 fully restored following local modi cation of chromatin with transiently expressed Gal4-p65 at a site near the promoter and no enhancement with the small molecule chromatin inhibitor UNC1999. These results, based on ectopic chromatin at a chromosomal transgene, implicate activator-mediated chromatin remodeling as an e ective method to enhance Cas9 editing at closed chromatin.