engleski

Development of epigenetic CRISPR-Cas9 system for targeted methylation at specific CpG sites

DNA methylation is an important epigenetic mechanism involved in gene regulation. In mammals, DNA methylation mostly occurs at symmetrical CpG dinucleotides. Cytosine methylation of gene regulatory regions has usually been associated with gene silencing, but functional studies have been limited due to the lack of methods for targeted manipulation of methylation marks. To that end, we developed a flexible, easily programmable tool for targeted methylation at specific CpG sites in mammalian cells, based on the CRISPR-Cas9 system. We completely abolished the nuclease activity of Cas9 protein and added the DNA methyltransferase domain of human DNMT3A, using a short peptide linker. We validated this newly developed dCas9-DNMT3A fusion construct in HEK293 cells, where we targeted unmethylated promoter regions of two different genes, BACH2 and IL6ST. At both loci, we tested a number of targeting single guide RNAs (sgRNAs) binding in different orientations and at different positions relative to the CpG sites analysed by bisulphite pyrosequencing. In most cases, dCas9-DNMT3A fusion induced significant increase in methylation level at cytosines adjacent to sgRNA-binding site, reaching up to 30-60% at particular CpG sites. Transfection of pooled sgRNAs targeting the same locus induced even higher methylation increase and decrease in the gene transcript level. The dCas9-DNMT3A fusion protein can be used to efficiently introduce CpG methylation at specific genomic locus and to probe the function of specific CpG methylation events in gene regulation.

CRISPR-Cas9 ; epigenetics ; targeted CpG methylation

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