engleski

Targeted methylation of BACH2 promoter by CRISPR/Cas9-based DNA methyltransferase

Cytosine methylation is an important epigenetic mechanism of transcriptional regulation. There are numerous examples of correlation between DNA methylation and gene expression, but causes and consequences of particular methylation events are still poorly defined. The aim of our study was to develop and validate an easily programmable tool, based on the CRISPR/Cas9 system, for targeted methylation of specific cytosines in the region of interest. The nuclease activity of previously described Cas9 nickase was completely abolished and the deactivated version of Cas9 (dCas9) was fused to the catalytic domain of human DNMT3a using a short peptide linker. In order to validate dCas9-DNMT3a fusion construct, we targeted BACH2 promoter, which contains a CpG island unmethylated in HEK293 cell line. Eight different guide RNAs (gRNAs) were designed to direct the fusion protein to the complementary target sites in BACH2 promoter. After a short puromycin selection, DNA was isolated from transfected HEK293 cells and bisulphite converted. Methylation status of target region was analysed using pyrosequencing, and results showed a significant elevation in methylation level of cytosines adjacent to gRNA-binding site. Several guides resulted in 20-50% increase in CpG methylation level. The fusion protein comprising deactivated Cas9 nuclease and DNA methyltransferase domain efficiently catalysed CpG methylation at specific genomic locus in human cells. This newly developed tool can be used to examine the role of specific CpG methylation sites in the regulation of transcription.

Metyhlation ; CRISPR/Cas9-based DNA methyltransferase ; BACH2

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