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Protein engineering strategies for improving the selective methylation of target CpG sites by a dCas9-directed cytosine methyltransferase in bacteria.

Authors :
Xiong, Tina
Rohm, Dahlia
Workman, Rachael E.
Roundtree, Lauren
Novina, Carl D.
Timp, Winston
Ostermeier, Marc
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