854 CRISPR-Cas9-based epigenome editing targeting the CDKN2A promoter reactivates the p16INK4A tumor suppressor and inhibits proliferation of skin cancer cells

Inactivation of tumor suppressor genes is a frequent event in carcinogenesis. In skin cancer, the CDKN2A gene, which encodes p16INK4A and p14ARF proteins, often silenced by epigenetic abnormalities such as promoter DNA methylation histone deacetylation. The cell cycle regulator plays key role growth metastasis. Unlike gene loss, silencing aberrant modifications can be reversed small-molecule compounds methyltransferase inhibitors deacetylase inhibitors. However, these are nonspecific, affecting globally. Thus, we used CRISPR-Cas9-based epigenome editing tools for targeted acetylation at genomic locus. To induce promoter, nuclease-deactivated Cas9 (dCas9) was fused to catalytic domain acetyltransferase p300 so that dCas9-p300 fusion protein could recruited target site guide RNA (gRNA) bound promoter. After with gRNA targeting introduced into A431 cancer line, performed RT-qPCR revealed p16INK4AmRNA expression levels increased approximately 30-fold. We also proliferation assays found cells decreased 20%. fully inhibit cells, further investigations needed evaluate combined effects demethylation This study provides insight how novel order reactivate dormant suppressors phenotypes.