Histone modifications and active gene expression are associated with enhanced CRISPR activity in de-silenced chromatin

ABSTRACT Recently we demonstrated that closed chromatin composed of Polycomb proteins and histone 3 lysine 27 trimethylation impedes CRISPR-mediated genome editing by blocking the accessibility of chromosomal DNA to spCas9/sgRNA. Editing efficiencies were higher in cells where the same reporter locus had not been repressed, thus we presume that silenced chromatin can be modified to generate a Cas9-accessible state. To test this idea, we exposed the locus to antagonists of Polycomb silencing: Gal4-p65, a targeted transcriptional activator, and UNC1999, a chemical inhibitor of the histone H3K27 methyltransferase EZH2. For both we observed loss of histone trimethylation. Only Gal4-p65 treatment increased target gene expression. Initial Gal4-p65 overexpression impedes Cas9 activity, while a 9-day recovery period leads to enhanced Cas9 efficiency up to 1000 bp from the Gal4 binding site. No enhancement was observed with UNC1999. These results demonstrate the strong influence of transcription-driven chromatin remodeling on CRISPR editing at closed chromatin.