%0 Journal Article
%T Protocol for DNA Methylation Editing of Imprinted Loci and Assessment of the Effects.
%A Sekita Y
%A Kimura T
%J Methods Mol Biol
%V 2842
%N 0
%D 2024
%M 39012595
暂无%R 10.1007/978-1-0716-4051-7_8
%X In this chapter, we present an experimental protocol to conduct DNA methylation editing experiments, that is, to induce loss or gain of DNA methylation, targeting Dlk1-Dio3 imprinted domain, a well-studied imprinted locus, in ES cells. In this protocol, plasmid vectors expressing the DNA methylation editing tools, combining the CRISPR/dCas9 system and the SunTag system coupled to a DNA methyltransferase or a TET enzyme, are introduced into cells for transient expression. By employing this strategy, researchers can effectively investigate a distinct DNA methylation signature that has an impact on the imprinting status, including gene expression and histone modifications, across the entire domain. We also describe strategies for allele-specific quantitative analyses of DNA methylation, gene expression, and histone modifications and binding protein levels for assessing the imprinting state of the locus.