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• W2002402255 startingPage "e96931" @default.
• W2002402255 abstract "The ability to target DNA methylation toward a single, user-designated CpG site in vivo may have wide applicability for basic biological and biomedical research. A tool for targeting methylation toward single sites could be used to study the effects of individual methylation events on transcription, protein recruitment to DNA, and the dynamics of such epigenetic alterations. Although various tools for directing methylation to promoters exist, none offers the ability to localize methylation solely to a single CpG site. In our ongoing research to create such a tool, we have pursued a strategy employing artificially bifurcated DNA methyltransferases; each methyltransferase fragment is fused to zinc finger proteins with affinity for sequences flanking a targeted CpG site for methylation. We sought to improve the targeting of these enzymes by reducing the methyltransferase activity at non-targeted sites while maintaining high levels of activity at a targeted site. Here we demonstrate an in vitro directed evolution selection strategy to improve methyltransferase specificity and use it to optimize an engineered zinc finger methyltransferase derived from M.SssI. The unusual restriction enzyme McrBC is a key component of this strategy and is used to select against methyltransferases that methylate multiple sites on a plasmid. This strategy allowed us to quickly identify mutants with high levels of methylation at the target site (up to ∼80%) and nearly unobservable levels of methylation at a off-target sites (<1%), as assessed in E. coli. We also demonstrate that replacing the zinc finger domains with new zinc fingers redirects the methylation to a new target CpG site flanked by the corresponding zinc finger binding sequences." @default.
• W2002402255 created "2016-06-24" @default.
• W2002402255 creator A5016482797 @default.
• W2002402255 creator A5028421625 @default.
• W2002402255 date "2014-05-08" @default.
• W2002402255 modified "2023-09-26" @default.
• W2002402255 title "Directed Evolution of Improved Zinc Finger Methyltransferases" @default.
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• W2002402255 doi "https://doi.org/10.1371/journal.pone.0096931" @default.
• W2002402255 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/4014571" @default.
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• W2002402255 hasPublicationYear "2014" @default.
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