Inheritable Silencing of Endogenous Genes by Hit-and-Run Targeted Epigenetic Editing

Gene silencing is a powerful approach for interrogating gene function and holds promise for therapeutic applications. Here we repurpose the ERV silencing machinery of embryonic stem cells to stably silence genes in somatic cells by epigenetics. This was achieved by transiently expressing combinations of artificial transcriptional repressors that bind to and synergize at the target locus to instruct de novo DNA methylation, thus ensuing long-term memory of the repressive epigenetic state. Silencing was highly specific and sharply confined to the targeted gene, was resistant to activation induced by cytokine stimulation and could be relieved only by targeted DNA demethylation. We demonstrate the portability of this technology by silencing three highly transcribed genes, B2M, IFNAR1, and VEGFA, using multiple DNA binding platforms and interrogating thousands of genomic loci after reporter insertion in different cell types, including primary T-lymphocytes. Finally, we expanded the breadth of targeted epigenome editing by multiplex gene silencing, setting the stage for its application in research and medicine. Overall design: MeDIP-seq and RNA-seq profiling of K562 cells expressing B2M:tdTomato transgene and silenced by dCas9 or TALE directed DNA methylation targeting B2M promoter