Open AccessProtein engineering strategies for improving the selective methylation of target CpG sites by a dCas9-directed cytosine methyltransferase in bacteria
Author(s) -
Tina Xiong,
Dahlia Rohm,
Rachael E. Workman,
Lauren Roundtree,
Carl D. Novina,
Winston Timp,
Marc Ostermeier
Publication year - 2018
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0209408
Subject(s) - methylation , cpg site , methyltransferase , dna methylation , biology , dna methyltransferase , epigenetics of physical exercise , microbiology and biotechnology , nuclease , genetics , dna , chemistry , gene , gene expression
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.