Protein engineering strategies for improving the selective methylation of target CpG sites by a dCas9-directed cytosine methyltransferase in bacteria. Xiong, T. A. R., Dahlia AND Workman, R. E. A. R., Lauren AND Novina, C. D. A. T., & Winston AND Ostermeier, M. PLoS One, 13(12):1-18, Public Library of Science, 12, 2018. ![Protein engineering strategies for improving the selective methylation of target CpG sites by a dCas9-directed cytosine methyltransferase in bacteria [link]](https://bibbase.org/img/filetypes/link.svg "link")
Paper doi abstract bibtex 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.
@article{10.1371/journal.pone.0209408,
author = {Xiong, Tina AND Rohm, Dahlia AND Workman, Rachael E. AND Roundtree, Lauren AND Novina, Carl D. AND Timp, Winston AND Ostermeier, Marc},
journal = "PLoS One",
publisher = {Public Library of Science},
title = {Protein engineering strategies for improving the selective methylation of target {CpG} sites by a {dCas9-directed} cytosine methyltransferase in bacteria},
year = {2018},
month = {12},
volume = {13},
url = {https://doi.org/10.1371/journal.pone.0209408},
pages = {1-18},
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.},
number = {12},
doi = {10.1371/journal.pone.0209408},
author+an = {3=student; 6=pi}
}
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