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Open AccessArticle

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

Institute of Biochemistry and Technical Biochemistry, Department of Biochemistry, Stuttgart University, Allmandring 31, 70569 Stuttgart, Germany
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Authors to whom correspondence should be addressed.
These authors contributed equally to this work.
Int. J. Mol. Sci. 2020, 21(2), 502; https://doi.org/10.3390/ijms21020502
Received: 20 November 2019 / Revised: 7 January 2020 / Accepted: 8 January 2020 / Published: 13 January 2020
(This article belongs to the Special Issue Advances in Epigenome Editing)
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. View Full-Text
Keywords: epigenome editing; targeted DNA methylation; dCas9; SunTag; DNMT3A-DNMT3L epigenome editing; targeted DNA methylation; dCas9; SunTag; DNMT3A-DNMT3L
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MDPI and ACS Style

Hofacker, D.; Broche, J.; Laistner, L.; Adam, S.; Bashtrykov, P.; Jeltsch, A. Engineering of Effector Domains for Targeted DNA Methylation with Reduced Off-Target Effects. Int. J. Mol. Sci. 2020, 21, 502.

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