ArticlePloS one2014
Directed evolution of improved zinc finger methyltransferases.
Article in PloS one, 2014. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 18 papers.
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Who cites it
18 citing papers in PubMed, 30 citations in OpenAlex.
- Genome wide inherited modifications of the tomato epigenome by trans-activated bacterial CG methyltransferase.Cellular and molecular life sciences : CMLS · 2024Article
- Lowering DNA binding affinity of SssI DNA methyltransferase does not enhance the specificity of targeted DNA methylation in E. coli.Scientific reports · 2021Article
- Variations and expression features of CYP2D6 contribute to schizophrenia risk.Molecular psychiatry · 2021Article
- Editing DNA Methylation in Mammalian Embryos.International journal of molecular sciences · 2020Review
- DNA methylation and de-methylation using hybrid site-targeting proteins.Genome biology · 2018Review
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- Article
- Targeted DNA methylation in human cells using engineered dCas9-methyltransferases.Scientific reports · 2017Article
- Targeted DNA methylation in pericentromeres with genome editing-based artificial DNA methyltransferase.PloS one · 2017Article
- Modern Genome Editing Technologies in Huntington's Disease Research.Journal of Huntington's disease · 2017Review
- Re-expression of Selected Epigenetically Silenced Candidate Tumor Suppressor Genes in Cervical Cancer by TET2-directed Demethylation.Molecular therapy : the journal of the American Society of Gene Therapy · 2016Article
- Genome-wide analysis of DNA methylation and gene expression defines molecular characteristics of Crohn's disease-associated fibrosis.Clinical epigenetics · 2016Article
- Stable oncogenic silencing in vivo by programmable and targeted de novo DNA methylation in breast cancer.Oncogene · 2015Article
- Strategies for precision modulation of gene expression by epigenome editing: an overview.Epigenetics & chromatin · 2015Review
- Synthetic epigenetics-towards intelligent control of epigenetic states and cell identity.Clinical epigenetics · 2015Article
- Advances in the directed evolution of proteins.Current opinion in chemical biology · 2014Review
- Epigenetic mechanisms underlying the pathogenesis of neurogenetic diseases.Neurotherapeutics : the journal of the American Society for Experimental NeuroTherapeutics · 2014Review
- Environmental stressors and epigenetic control of the hypothalamic-pituitary-adrenal axis.Neuroendocrinology · 2014Review
Corrections and comments
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Authors and funding
2 authors at 1 institution in 1 country.
Funding
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.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.