Engineering epigenetic memory requires co-targeting of histone methylatransferases and DNA methylatransferases [Methylation]

Rewriting of the epigenome has risen as a promising alternative to gene editing for precision medicine. In nature, epigenetic silencing can result in complete attenuation of target gene expression over multiple mitotic divisions. However, persistent repression has been difficult to achieve using targeted systems. Here, we report that robust and persistent epigenetic memory required both a DNA methyltransferase (DNMT3A-dCas9) and a histone methyltransferase (Ezh2-dCas9 or KRAB-dCas9). DNMT3A-dCas9 was dependent on full-length DNMT3L for maximal activity. Co-targeting with Ezh2-dCas9, DNMT3A-dCas9 and DNMT3L induced long-term HER2 repression over at least 50 days and maintained a heterochromatic environment. Interestingly, substitution of Ezh2-dCas9 for KRAB-dCas9 enabled long-term repression at some target genes (e.g., SNURF) but not at HER2, at which H3K9me3 and DNA methylation were transiently acquired and subsequently lost. Off-target DNA hypermethylation occurred at many individual CpG sites but rarely at multiple CpGs in a single promoter, consistent with no detectable effect on transcription at the loci tested. Conversely, robust hypermethylation was observed at HER2. These data demonstrate that targeting different combinations of histone and DNA methyltransferases is required to achieve maximal repression at different loci in the same cell, or the same locus in different cells. Genome-wide nethylation analysis of cells after treatment with Ezh2-dCas9 or KRAB-dCas9 cocktail with DNMT3A/DNMT3L or with dCas9 control using the Infinium Human MethylationEPIC BeadChip (Illumina)