Optimized Protocol for the Regulation of DNA Methylation and Gene Expression Using Modified dCas9-SunTag Platforms
Sumiyo Morita1, Takuro Horii1, Izuho Hatada2
1Biosignal Genome Resource Center, IMCR, Gunma University, Maebashi, Japan.
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DNA methylation, one of the most studied epigenetic modifications, regulates many biological processes. Dysregulation of DNA methylation is implicated in the etiology of several diseases, such as cancer and imprinting diseases. Accordingly, technologies designed to manipulate DNA methylation at specific loci are considered worthwhile and many epigenome editing technologies have been developed, which were based on ZF, TALE, and CRISPR-dCas9. Here, we describe a protocol for the application of a modified dCas9-SunTag system, which increased the efficiency of targeted demethylation and gene activation at specific DNA loci. The original SunTag system consists of 10 copies of the GCN4 peptide separated by 5-amino-acid linkers. To achieve more efficient recruitment of an anti-GCN4 scFv fused to the ten-eleven (TET) 1 hydroxylase, an enzyme that demethylates DNA, we changed the linker length to 22 amino acids. Moreover, we describe the co-recruitment of TET1 and VP64 for efficient gene activation. Since we showed the manipulation of DNA methylation at specific loci and gene activation, its application could lead to its future use in the clinic.
