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

Carl D Novina,Marc Ostermeier,Dahlia Rohm,Albert Jeltsch,Tina Xiong,Lauren Roundtree,Winston Timp,Rachael E Workman

doi:10.1371/journal.pone.0209408

Carl D Novina, Marc Ostermeier + Show 6 more

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https://doi.org/10.1371/journal.pone.0209408

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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.

Highlights

Cytosine methylation is important in facilitating many mammalian biological processes such as chromosomal stability, genomic imprinting, X-chromosome inactivation and gene expression [1,2,3]
We have demonstrated the targeting abilities of deactivated Cas9 (dCas9) fused to a M.SssI split methyltransferase [13]–a split M.SssI that had been used previously in the context of zinc finger instead of dCas9 [19, 22]
Our findings offer insights and design rules for improving methylation as targeted by dCas9-MC/MN in E. coli

Summary

Introduction

Cytosine methylation is important in facilitating many mammalian biological processes such as chromosomal stability, genomic imprinting, X-chromosome inactivation and gene expression [1,2,3]. Methylation has been implicated in embryonic development and cell differentiation [1, 3,4,5]. High levels of methylation in promoter regions often lead to transcriptional silencing, but many questions remain about the mechanisms by which DNA methylation alters gene expression. The ability to modulate methylation in a targeted way will facilitate the understanding of CpG methylation and its functional role in the context of specific cis-. Improvements in the methylation selectivity of a dCas9-directed cytosine methyltransferase study design, data collection and analysis, decision to publish, or preparation of the manuscript

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