Engineering of effector domains for targeted DNA methylation with reduced off-target effects

Epigenome editing is a promising technology, potentially allowing the stable reprogramming of gene expression profiles without alteration of the DNA sequence. Targeted DNA methylation has been successfully documented by many groups for silencing of selected genes, but recent publications have raised concerns regarding its specificity. In the current work, we developed new EpiEditors for programmable DNA methylation in cells with a high efficiency and improved specificity. First, we demonstrated that the dCas9-SunTag scaffold, which has been used earlier for signal amplification, can be combined with the DNMT3A-DNMT3L effector domain allowing a strong methylation at the target genomic locus. We demonstrated that off-target activity of this system is mainly due to untargeted freely diffusing DNMT3A-DNMT3L subunits. Therefore, we generated several DNMT3A variants containing mutations, which reduced their endogenous DNA binding strength. We analyzed the genome-wide DNA methylation of selected variants and confirmed a striking reduction of untargeted methylation. For all potential applications of targeted DNA methylation, the efficiency and specificity of the treatment are the key factors. By developing highly active targeted methylation systems with strongly improved specificity, our work contributes to the future applications of this approach. HEK293 cells were transiently transfected with dCas9-SunTag, antibody-fused catalytic domain of DNMT3A/3L (wild type / DNMT3A mutants) and sgRNA targeting the CpG island of the ISG15 gene. After 3 days, the cells were sorted using FACS to obtain only cells carrying each plasmid. MBD2 pulldown was perfomed with sonicated genomic DNA and methylation changes were analysed genome-wide by NGS.