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Protein engineering strategies for improving the selective methylation of target CpG sites by a dCas9-directed cytosine methyltransferase in bacteria.
- Source :
-
PLoS ONE . 12/18/2018, Vol. 13 Issue 12, p1-18. 18p. - Publication Year :
- 2018
-
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. [ABSTRACT FROM AUTHOR]
Details
- Language :
- English
- ISSN :
- 19326203
- Volume :
- 13
- Issue :
- 12
- Database :
- Academic Search Index
- Journal :
- PLoS ONE
- Publication Type :
- Academic Journal
- Accession number :
- 133613170
- Full Text :
- https://doi.org/10.1371/journal.pone.0209408