(228ej) Site-Specific DNA Methylation Using Engineered dCas9-Methyltransferases

Heritable changes in gene expressions via epigenetic modifications are paramount to the normal functioning of the host and its phenotype. In particular, cytosine methylation has been shown to regulate gene expression in eukaryotes to facilitate vital cellular processes such as cellular differentiation and embryonic development. High levels of methylation at promoter regions are often associated with transcriptional silencing. One can thus envision that a bio-tool for promoter- specific methylation may aid in gene repression of overexpressed oncogenes or disease-linked genes during malignancy, such as leukemia, atherosclerosis, and Alzheimerâ??s disease The typical strategy for targeting DNA methylation involves end-to-end fusion of a DNA binding domain (DBD), such as zinc fingers and TALE, to a cytosine DNA methyltransferases (MTase), typically bacterial enzymes or human DNMT3a. The two main drawbacks of this approach are (1) methylation occurs at non-target sites since the MTase domain remains active to methylate these sites when the DBD is not interacting with DNA and (2) new sequence recognition requires DBD redesign. Our solution to the first problem is to use the target site for methylation as a template for assembling the active form of an otherwise ineffective split methyltransferase (sMTase). This strategy has been shown to result in very specific targeting in bacteria (~80% methylation at the target site and â?¤0.8% methylation at other CpG sites) using zinc fingers as the DBD. The second problem can be addressed by the CRISPR-Cas9 genome engineering tool. A key feature of nuclease- null Cas9 (dCas9) as a DBD is its ability to bind specific sites defined by the single-stranded guide RNA (sgRNA) sequence. A carefully selected sgRNA allows for highly specific targeting without the requirement for DBD redesign. An ideal dCas9-sMTase would have many exemplary features: the precision of true single-site targeting of methylation, ease of targeting via simple design of sgRNA, and multiplexed targeting of several sites via multiple sgRNAs. The later advantage addresses the concern that single site methylation might not be sufficient to have a desired phenotypic effect. To this end, we designed fusion proteins of dCas9 and a sMTase. The dCas9-sMTase provides high precision and control over CpG methylation in bacteria and human cells.