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

Autor: Tina Xiong, Dahlia Rohm, Rachael E Workman, Lauren Roundtree, Carl D Novina, Winston Timp, Marc Ostermeier
Jazyk: angličtina
Rok vydání: 2018
Předmět:
Zdroj: PLoS ONE, Vol 13, Iss 12, p e0209408 (2018)
Druh dokumentu: article
ISSN: 1932-6203
DOI: 10.1371/journal.pone.0209408
Popis: 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.
Databáze: Directory of Open Access Journals
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