engleski

Targeted CpG methylation using the modified CRISPR-Cas9 system

We have repurposed the CRISPR-Cas9 system for DNA methylation at targeted CpG sites. This novel tool enables epigenetic silencing of gene expression by methylation of CpG sites in regulatory regions of targeted genes. 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 to that, epigenetic editing at specific sites in the genome could selectively alter the gene expression pattern. The novel tool for specific DNA methylation that we developed consists of deactivated Cas9 (dCas9) nuclease fused to the catalytic domain of the DNA methyltransferase DNMT3A. It is targeted to any 20 bp sequence followed by the NGG trinucleotide by co expression of a guide RNA. We demonstrated CpG methylation by the fusion protein in a ~35 bp wide region. We also showed that multiple guide RNAs could target the dCas9-DNMT3A construct to multiple sites, which enabled methylation of a wider genome region. Alternatively, multiple targeting could be used to silence several genes simultaneously. DNA methylation activity was highly specific for the targeted region and heritable across mitotic divisions. Finally, we demonstrated that directed DNA methylation of the wider region in the promoter of the target locus IL6ST decreased its expression, which served as a proof of the concept of artificial epigenetic silencing by targeted CpG methylation in vivo.

Epigenome Editing ; CpG Methylation ; Cas9 ; CRISPR ; DNMT3A

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