engleski

Modulation of glyco-gene expression using CRISPR/Cas9-based molecular tools for epigenetic editing

Gene expression can be regulated by epigenetic mechanisms, such as DNA methylation at CpG dinucleotides in gene promotes. This is particularly important for glyco-genes, since protein N-glycosylation seems to be epigenetically regulated. We have constructed molecular tools for targeted DNA methylation and demethylation by repurposing the CRISPR- Cas9 system. The Cas9 protein can be targeted to almost any 20 bp sequence by co-expression of a guide RNA. To facilitate targeted CpG methylation, catalytic domain of the DNA methyltransferase DNMT3A was fused to catalytically inactive Cas9 (dCas9). This fusion enabled silencing of gene expression by methylation of CpG sites in regulatory regions of the targeted genes – the BACH2 and IL6ST, involved in IgG glycosylation. Targeted gene promoter demethylation was accomplished by an analogous construct comprising dCas9-TET1 catalytic domain fusion, validated in the MGAT3 and LAMB1 gene promoters that are also involved in IgG glycosylation. Rigorous characterisation of our constructs in the HEK293 cell line showed peak methylation and demethylation activity about 30 nucleotides downstream from the targeted site within a ~35 bp wide window. Changes in methylation and demethylation at the target were up to 60%, with values around 30% being typical. Activity was highly specific for targeted regions and heritable across cell divisions. Multiple guide RNAs could target the dCas9-DNMT3A construct to multiple sites, which enabled hypermethylation of a wider genome region. We demonstrated that the 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 reversible nature of epigenetic modifications, including DNA methylation, has been already exploited in cancer therapy for remodelling the aberrant epigenetic landscape. Epigenetic editing at specific sites could alter the gene expression pattern selectively, unlike the widely used epigenetic inhibitors. Thus, our system represents promising molecular tools for unravelling details of molecular processes in a living cell, especially protein glycosylation. Development of a comprehensive regulatory toolbox is underway, with potential uses in gene therapy and glyco-engineering.

CRISPR-Cas9 ; DNMT3A ; TET1 ; epigenetics ; methylation

DOI: 10.1007/s10719-017-9784-5