234: Programmable epigenetic silencing of enhancers with CRISPR based epigenetic modifiers

Abstract: The transcriptional start site and enhancers of expressed genes are decorated with active epigenetic marks (i.e. H3K27ac and H3K4me3), highlighting the critical role of epigenetic mechanisms in regulating these non-coding genomic elements. The CRISPR based epigenetic editor CRISPRi induces strong gene silencing by depositing H3K9me3 therefore inducing gene suppression. CRISPRi has been used to study enhancer regions; however, other histone modifications may be necessary to silence enhancer regions and study the biology and function of enhancers. To address this, I targeted five epigenome editors: MeCP2, RCOR1, SAP25, dCas9, and CRISPRi to modify chromatin structure and evaluate gene silencing at enhancer regions. Each editor functions uniquely to repress gene expression, with the main difference being MeCP2, SAP25, and RCOR1 recruit various repressor complexes that deacetylates H3k27ac. By engineering single guide RNAs, we are able to target the enhancer regions of essential genes: GATA1, MYC, and HDAC6, within K562 cell lines which constitutively express dCas9 fusions. To test for effects on cell proliferation, we stably expressed sgRNAs targeting essential or non-essential genes in our parental cell lines through lentiviral transduction, followed by FACS sorting. We then monitored cell proliferation over 12 days using an internally normalized competitive growth assay and showed that: enhancer targeting cell lines produced intermediate silencing compared to cells that were targeted at the transcriptional start site (TSS) and CRISPRi continues to be the most functional way to silence enhancer regions. This experiment contributes to our understanding of enhancer regions and demonstrates how alternative epigenome editors provide distinct approaches for enhancer silencing and therapeutic targeting.