Editing of DNA methylation using dCas9-peptide repeat and scFv-TET1 catalytic domain fusions

S Morita, T Horii, I Hatada - Epigenome Editing: Methods and Protocols, 2018 - Springer
S Morita, T Horii, I Hatada
Epigenome Editing: Methods and Protocols, 2018Springer
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 …
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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