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

Abstract

Targeted DNA methylation is a technique that aims to methylate cytosines in selected genomic loci. In the most widely used approach a CG-specific DNA methyltransferase (MTase) is fused to a sequence specific DNA binding protein, which binds in the vicinity of the targeted CG site(s). Although the technique has high potential for studying the role of DNA methylation in higher eukaryotes, its usefulness is hampered by insufficient methylation specificity. One of the approaches proposed to suppress methylation at unwanted sites is to use MTase variants with reduced DNA binding affinity. In this work we investigated how methylation specificity of chimeric MTases containing variants of the CG-specific prokaryotic MTase M.SssI fused to zinc finger or dCas9 targeting domains is influenced by mutations affecting catalytic activity and/or DNA binding affinity of the MTase domain. Specificity of targeted DNA methylation was assayed in E. coli harboring a plasmid with the target site. Digestions of the isolated plasmids with methylation sensitive restriction enzymes revealed that specificity of targeted DNA methylation was dependent on the activity but not on the DNA binding affinity of the MTase. These results have implications for the design of strategies of targeted DNA methylation.

Genome wide inherited modifications of the tomato epigenome by trans-activated bacterial CG methyltransferase

“…M.SssI from the Mollicutes spiroplasma species is a bacterial methylase that catalyzes specifically CG methylation [ 40 ]. M.SssI was shown to be active in vitro, associated with Zinc Finger (ZF) proteins [ 4 , 54 ], triple-helix-forming oligonucleotides [ 48 ] or catalytically-inactive Cas9 (dCas9) [ 28 ] and in vivo with dCas9 in E. coli [ 42 , 53 ], mammalian cells [ 52 ], mouse oocytes or embryos [ 55 ] or with Transcription Activator-Like Effector (TALE) fusion proteins in mouse [ 56 ]. The ability of different M.SssI variants fused to a dCas9 to induce methylation in a specific locus was also demonstrated in Arabidopsis [ 13 ], as well as the potential of the newly acquired methylation to be inherited .…”

Increasing Specificity of Targeted DNA Methylation Editing by Non-Enzymatic CRISPR/dCas9-Based Steric Hindrance

“…For M.SssI (Q147L), the authors analyzed candidate gRNA off-target sites of the gRNA and reported no off-target methylation-yet, this off-target analysis strategy is insufficient in that it does not assess the aforementioned off-target methylation independent of dCas9 by the methyltransferase domain as has been extensively demonstrated. Though the authors also performed reduced-representation bisulfite sequencing, they correctly conclude that a failure to detect off-target effects with these approaches does not equate to an absence of off-target effects and, indeed, a more recent study presented evidence that the Q147L mutation in M.SssI does not reduce its nonspecific activity [48], instead only reducing its catalytic activity which suggests that in the original study, the Q147L mutation leads to off-target methylation that is below the threshold of detection. Off-target detection is highly dependent on the power and comprehensiveness of the detection method; it is likely that deep whole-genome methylation sequencing-which was not used in either study-would reveal the true extent of off-target methylation of these engineered epigenetic editor variants.…”

Microbial Synthetic Epigenetic Tools Design and Applications

“…To address issues of leaky gene expression, the specificity of zinc finger protein binding and transferase activity can be fine-tuned. For example, when epigenetic modifying proteins are fused with Cas9’s targeting capabilities, they offer novel avenues for precision gene regulation (Figure C).…”