3rd International Conference on CRISPR Technologies

Inhibition and Recovery of CRISPR/spCas9 Activity at Closed Chromatin in a Human Cell Line

Authors

Haynes, K. - Presenter, Emory University
Daer, R., Arizona State University
Hamna, F., Arizona State University
Barrett, C., Arizona State University
To efficiently and reliably edit human DNA in living cells with CRISPR, the Cas9/gRNA complex must overcome barriers imposed by chromatin-mediated compaction. Chromatin is a system of proteins and nucleic acids that organize genomic DNA within the nuclei of eukaryotic cells. Chromatin composed of Polycomb-family proteins mediates epigenetic repression in stem cells and differentiated cells, and is distributed throughout the genome at hundreds of sites that drive cell development pathways. Therefore biomedically important target genes may often be inaccessible to CRISPR. Previously, we demonstrated that closed, silenced chromatin impedes CRISPR editing by making the DNA less accessible to Cas9/gRNA binding. Our investigation of the underlying mechanism showed that disruption of chromatin through siRNA-mediated knock-down of the Polycomb complex enhanced CRISPR editing. Interestingly, target gene overexpression also impeded CRISPR activity. To further investigate the requirements for inducing a CRISPR-accessible state, we compared two approaches: a broad-acting Polycomb-disrupting drug (EZH2 inhibitor UNC1999) and site-specific chromatin-modifying fusion proteins. We explored the mechanism of enhanced editing at artificially opened chromatin in HEK293 cells using deep sequencing of edited DNA and chromatin immunoprecipitation (ChIP) of histone modifications. We demonstrate strong CRISPR enhancement with transient expression of the site-specific, fusion transcriptional activator Gal4P65 and no enhancement with the small molecule chromatin inhibitor UNC1999. In both cases H3K27me3 was depleted from the CRISPR target site, but only Gal4P65 gene-activating enhanced CRISPR efficiency. CRISPR activity was also restored by other Gal4 fusions that did not activate luciferase expression. Our results support the use of site-specific, activator-associated fusion proteins as an effective method to enhance Cas9 editing within Polycomb-repressed chromatin.