biostudies-literatureXiong TNIDDK NIH HHSNational Institutes of HealthNational Science Foundatione0209408https://www.ebi.ac.uk/biostudies/studies/S-EPMC6298699biostudies-literatureUnknown13(12)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.PloS oneProtein engineering strategies for improving the selective methylation of target CpG sites by a dCas9-directed cytosine methyltransferase in bacteria.PMC6298699CBET-1505793CBET-15106521DP1 DK105602-01DP1 DK105602Roundtree LWorkman RENovina CDXiong TRohm DOstermeier MTimp WfalseProtein engineering strategies for improving the selective methylation of target CpG sites by a dCas9-directed cytosine methyltransferase in bacteria.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.2018-01-01T00:00:00Z20182024-12-03T21:16:55.275Z2019-03-26T22:28:49ZS-EPMC62986993056238810.1371/journal.pone.0209408