engleski

CRISPR/Cas9-based molecular tools for epigenetic manipulations

We have repurposed the CRISPR-Cas9 system for targeted DNA methylation and demethylation. De novo DNA methyltransferase DNMT3A was fused to catalytically inactive Cas9 (dCas9) and this fusion enabled silencing of gene expression by methylation of CpG sites located in regulatory regions of the targeted candidate genes - the BACH2 and IL6ST, involved in IgG glycosylation. Targeted gene promoter demethylation was accomplished by an analogous construct comprising dCas9-TET1 fusion and the tool activity was validated on the MGAT3 and LAMB1 gene promoters involved in IgG glycosylation, as well. We set out to rigorously characterize the activity of our constructs in a HEK293 model cell line. The dCas9- DNMT3 tool can be targeted to any 20 bp sequence followed by the NGG trinucleotide by co expression of a guide RNA, with the peak activity throughout a ~35 bp wide region. DNA methylation activity was highly specific for the targeted region and heritable across mitotic divisions. We also showed that multiple guide RNAs could target the dCas9-DNMT3A construct to multiple sites, which enabled hypermethylation of a wider genome region. We demonstrated that the candidate gene IL6ST decreased expression level following promoter hypermethylation, which served as a proof of the concept of artificial epigenetic silencing by targeted CpG methylation in vivo. The TET1-based molecular tool for targeted demethylation showed similar activity pattern to Cas9-DNMT3A fusion: up to 60% demethylation and the peak activity about 30 bp downstream from the binding site. The reversible nature of epigenetic modifications, including DNA methylation, has been already exploited in cancer therapy for remodelling the aberrant epigenetic landscape. However, the classical approach uses epigenetic inhibitors non-selectively. In contrast, epigenetic editing at specific sites could selectively alter the gene expression pattern. Thus, our newly developed constructs represent promising molecular tools for unravelling details of molecular processes in a living cell (such as protein glycosylation), and they have potential as therapeutics upon further development.

CRISPR/Cas9 ; DNMT3a ; TET1 ; epigenetics ; DNA methylation

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