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Epigenome editing has rapidly evolved in recent years, with diverse applications that include elucidating gene regulation mechanisms, annotating coding and noncoding genome functions and programming cell state and lineage specification. Importantly, given the ubiquitous role of epigenetics in complex phenotypes, epigenome editing has unique potential to impact a broad spectrum of diseases. By leveraging powerful DNA-targeting technologies, such as CRISPR, epigenome editing exploits the heritable and reversible mechanisms of epigenetics to alter gene expression without introducing DNA breaks, inducing DNA damage or relying on DNA repair pathways. Epigenome editing has versatile applications in biomedical research and disease treatment. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]