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.
Highlights
The targeted alteration of gene expression is a widely used approach in basic research as well as in the development of new therapeutic strategies
In the case of deactivated Cas9 protein (dCas9)-DNMT3A, recruitment of four molecules is required to generate one catalytically active tetramer (Figure 1B), while dCas9 fused to DNMT3A-DNMT3L needs only to dimerize to form an active complex (Figure 1C)
The dCas9-SunTag provides a scaffold for the recruitment of several chimeric scFv-GCN4-DNMT3A-DNMT3L (Ab-3A3L) proteins with two potential mechanisms of assembly (Figure 1D)
Summary
The targeted alteration of gene expression is a widely used approach in basic research as well as in the development of new therapeutic strategies. Tuning of gene expression can be achieved by modulation of epigenomic signals, which regulate transcription at gene control elements, mainly promoters. This fundamental difference in the mechanism of action from the predecessors gives rise to three key advantages: established expression profiles can be stably maintained by cellular epigenetic machinery; they are reversible; and the method does not change the genetic information. The key instruments for epigenome editing are the so-called EpiEditors, artificial proteins constructed from two functional units, one of which is responsible for the delivery of the construct to the targeted genomic region and the other for the local modification of the chromatin state to establish the desired properties. To produce a functional DNA binding unit, dCas needs to form a complex with a single guide RNA (sgRNA), which is designed to contain a sequence complementary to the targeted genomic locus next to a protospacer adjacent motif site (Figure 1A) [6–8]
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