Engineering of Effector Domains for Targeted DNA Methylation with Reduced Off-Target Effects

Abstract

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 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 catalytically deactivated Cas9 protein (dCas9)-SunTag scaffold, which has been used earlier for signal amplification, can be combined with the DNMT3A-DNMT3L single-chain effector domain, allowing for 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-DNMT3L variants containing mutations in the DNMT3A part, which reduced their endogenous DNA binding. We analyzed the genome-wide DNA methylation of selected variants and confirmed a striking reduction of untargeted methylation, most pronounced for the R887E mutant. 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 future applications of this approach.

Lowering DNA binding affinity of SssI DNA methyltransferase does not enhance the specificity of targeted DNA methylation in E. coli

“…The specificity of targeted DNA methylation in E. coli was found to be strongly dependent on the intracellular MTase activity, but reducing the DNA binding affinity of the MTase domain had little if any influence on methylation specificity. These results shed new light on data obtained with mutant DNA MTases, where increased specificity of targeted DNA methylation was attributed to the weakened DNA binding affinity of the MTase 20 , 21 , 23 , 24 , 26 , 38 .…”

“…This work was started with the assumption that the preference of a chimeric MTase for the target site vs non-target sites could be increased by reducing the binding strength between the DNA and the MTase. In this model, the overall low substrate binding affinity of the mutant MTase is, at the addressed site, compensated by the increased effective local concentrations of the substrate site and the tethered MTase 20 , 21 , 23 , 24 , 38 .…”

“…The approaches of targeted DNA methylation share the basic principle of the pioneering study 7 : a CG-specific DNA MTase is linked to a targeting domain, which guides and anchors the MTase to the intended genomic site enabling preferential methylation of closely located CG sites (for recent reviews see 8 – 10 ). Most approaches to targeted DNA methylation used the de novo mammalian DNA MTase Dnmt3A (catalytic domain alone or in fusion with Dnmt3L) 11 – 20 or the CG-specific bacterial DNA MTase M.SssI 7 , 21 – 25 . Former studies used zinc finger (ZF) proteins 7 , 11 , 26 – 29 or Transcription Activator-like Effector (TALE) proteins 25 , 30 as targeting modules.…”

“…Former studies used zinc finger (ZF) proteins 7 , 11 , 26 – 29 or Transcription Activator-like Effector (TALE) proteins 25 , 30 as targeting modules. Zinc finger and TALE-mediated targeting of DNA methylation has recently been replaced by CRISPR-dCas9-guided targeting, which provides much greater flexibility 12 , 14 – 18 , 20 , 25 .…”

Reprogramming the anti-tumor immune response via CRISPR genetic and epigenetic editing

“…The dCas9-SunTag is based on single-chain variable fragment antibodies and the corresponding epitope, which offers major advantages, including high affinity and recognition of short peptide sequences. This system has previously been adapted to recruit DNA methyltransferases, including DNMT3A 63 and DNMT3L, 64 in order to induce locus-specific repression via DNA methylation, with minimal off-target binding. 65 Importantly, these next-generation dCas9 systems (SAM and SunTag) provide highly specific, effective, and tunable tools for targeted epigenetic manipulation.…”