Synthetic Readers and Writers of Chromatin to Advance Cell Engineering

Our group leverages the rich biological information encoded in nuclear proteins, collectively known as chromatin complexes, to manipulate gene expression states and DNA accessibility in mammalian cells. We have designed synthetic “readers” to interact with specific post-translational modifications (PTMs) on DNA-bound histone proteins to reactivate dormant tumor suppressor genes in cancer cells (Haynes & Silver 2011, Nyer et al. 2017, Olney et al. 2017). We and others have shown that synthetic “writers” can be used add or remove histone PTMs to control the local, physical state of chromatin packaging. Recently, we demonstrated that Polycomb-group proteins that regulate developmental genes in stem and cancer cells support a closed, silenced chromatin state that impedes CRISPR editing by reducing accessibility of DNA to Cas9/gRNA (Daer et al. 2016). siRNA-mediated knock-down of Polycomb enhanced CRISPR-mediated editing. Encouraged by this result, we set out to establish chromatin manipulation as a general, practical tool to improve gene editing. Here, we compare two methods for inducing an open, non-silenced state to support CRISPR efficiency: chromatin-disrupting inhibitor drugs and transient expression of site-specific chromatin-modifying 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 observed strong CRISPR enhancement with transient expression of the site-specific, fusion transcriptional activator Gal4-p65 but no significant enhancement with the small molecule chromatin inhibitor UNC1999. Loss of H3K27me3, gain of H3K4me3, and increased gene expression were associated with the CRISPR-permissive state. These results establish an effective method for enhancing CRISPR activity through the artificial regulation of gene expression and chromatin states in cultured mammalian cells.