Abstract
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 very important, and many epigenome editing technologies have been developed, based on zinc finger proteins, TALEs, and CRISPR/dCas9 targeting. We describe a protocol to induce and assess DNA demethylation on a target gene. It is based on a modification of the dCas9-SunTag system for efficient, targeted demethylation at specific DNA loci. The original SunTag system consists of ten copies of the GCN4 peptide separated by 5-amino-acid linkers. To achieve 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.
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Acknowledgments
This work was supported by the Basic Science and Platform Technology Program for Innovative Biological Medicine from the Ministry of Education, Culture, Sports, Science and Technology, Japan (MEXT), and the Japan Agency for Medical Research and Development (AMED).
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Morita, S., Horii, T., Hatada, I. (2018). Editing of DNA Methylation Using dCas9-Peptide Repeat and scFv-TET1 Catalytic Domain Fusions. In: Jeltsch, A., Rots, M. (eds) Epigenome Editing. Methods in Molecular Biology, vol 1767. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7774-1_23
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DOI: https://doi.org/10.1007/978-1-4939-7774-1_23
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