Laboratory of Stem Cell Biology, Department of Biosciences, Kitasato University School of Science, Sagamihara, Kanagawa, Japan.
Methods Mol Biol. 2024;2842:167-178. doi: 10.1007/978-1-0716-4051-7_8.
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.
在本章中，我们介绍了一个实验方案，用于进行 DNA 甲基化编辑实验，即诱导 Dlk1-Dio3 印迹域的 DNA 甲基化丢失或获得，这是一个经过充分研究的印迹基因座。在该方案中，表达 DNA 甲基化编辑工具的质粒载体被引入细胞进行瞬时表达，这些工具结合了 CRISPR/dCas9 系统和 SunTag 系统，与 DNA 甲基转移酶或 TET 酶偶联。通过采用这种策略，研究人员可以有效地研究整个基因座中对印迹状态有影响的特定 DNA 甲基化特征，包括基因表达和组蛋白修饰。我们还描述了用于等位基因特异性定量分析 DNA 甲基化、基因表达和组蛋白修饰以及结合蛋白水平的策略，以评估该基因座的印迹状态。
