ArticlePloS one2018
Protein engineering strategies for improving the selective methylation of target CpG sites by a dCas9-directed cytosine methyltransferase in bacteria.
Article in PloS one, 2018. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 6 papers.
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Who cites it
6 citing papers in PubMed, 14 citations in OpenAlex.
- CRISPR-based functional genomics tools in vertebrate models.Experimental & molecular medicine · 2025Review
- Genome wide inherited modifications of the tomato epigenome by trans-activated bacterial CG methyltransferase.Cellular and molecular life sciences : CMLS · 2024Article
- Reshaping the Landscape of the Genome: Toolkits for Precise DNA Methylation Manipulation and Beyond.JACS Au · 2024Review
- Lowering DNA binding affinity of SssI DNA methyltransferase does not enhance the specificity of targeted DNA methylation in E. coli.Scientific reports · 2021Article
- Anatomy of noncovalent interactions between the nucleobases or ribose and π-containing amino acids in RNA-protein complexes.Nucleic acids research · 2021Article
- Engineering of Effector Domains for Targeted DNA Methylation with Reduced Off-Target Effects.International journal of molecular sciences · 2020Article
Corrections and comments
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Authors and funding
7 authors at 2 institutions in 1 country.
Funding
Abstract
Mammalian gene expression is a complex process regulated in part by CpG methylation. The ability to target methylation for de novo gene regulation could have therapeutic and research applications. We have previously developed a dCas9-MC/MN protein for targeting CpG methylation. dCas9-MC/MN is composed of an artificially split M.SssI methyltransferase (MC/MN), with the MC fragment fused to a nuclease-null CRISPR/Cas9 (dCas9). Guide RNAs directed dCas9-MC/MN to methylate target sites in E. coli and human cells but also caused some low-level off-target methylation. Here, in E. coli, we show that shortening the dCas9-MC linker increases methylation of CpG sites located at select distances from the dCas9 binding site. Although a shortened linker decreased methylation of other CpGs proximal to the target site, it did not reduce off-target methylation of more distant CpG sites. Instead, targeted mutagenesis of the methyltransferase's DNA binding domain, designed to reduce DNA affinity, significantly and preferentially reduced methylation of such sites.
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Registered trials
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