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

• DOI: 10.1371/journal.pone.0209408
• Publication Date: 2018-12-18T18:26:39Z
• Authors: Tina Xiong; Dahlia Rohm; Rachael E. Workman; Lauren Roundtree; Carl D. Novina; Winston Timp; Marc Ostermeier
• Keywords: CpG site; DNA methyltransferase; Nuclease

Mammalian gene expression is a complex process regulated in part by CpG methylation. The ability to target methylation for de novo regulation could have therapeutic and research applications. We previously developed dCas9-MC/MN protein targeting composed of an artificially split M.SssI methyltransferase (MC/MN), with the MC fragment fused nuclease-null CRISPR/Cas9 (dCas9). Guide RNAs directed methylate sites E. coli human cells but also caused some low-level off-target Here, coli, we show that shortening dCas9-MC linker increases located at select distances from dCas9 binding site. Although shortened decreased other CpGs proximal site, it did not reduce more distant sites. Instead, targeted mutagenesis methyltransferase's DNA domain, designed affinity, significantly preferentially reduced such