Institute of Biochemistry and Technical Biochemistry, Department of Biochemistry, University of Stuttgart, Stuttgart, Germany.
Methods Mol Biol. 2023;2577:177-188. doi: 10.1007/978-1-0716-2724-2_12.
Epigenome editing is a powerful approach for the establishment of a chromatin environment with desired properties at a selected genomic locus, which is used to influence the transcription of target genes and to study properties and functions of gene regulatory elements. Targeted DNA methylation is one of the most often used types of epigenome editing, which typically aims for gene silencing by methylation of gene promoters. Here, we describe the design principles of EpiEditors for targeted DNA methylation and provide step-by-step guidelines for the realization of this approach. We focus on the dCas9 protein as the state-of-the-art DNA targeting module fused to 10×SunTag as the most frequently used system for editing enhancement. Further, we discuss different flavors of DNA methyltransferase modules used for this purpose including the most specific variants developed recently. Finally, we explain the principles of gRNA selection, outline the setup of the cell culture experiments, and briefly introduce the available options for the downstream DNA methylation data analysis.
表观基因组编辑是一种在选定的基因组位点上建立具有所需特性的染色质环境的强大方法，用于影响靶基因的转录，并研究基因调控元件的性质和功能。靶向 DNA 甲基化是最常用的表观基因组编辑类型之一，通常旨在通过基因启动子的甲基化来沉默基因。在这里，我们描述了用于靶向 DNA 甲基化的 EpiEditors 的设计原则，并提供了实现该方法的逐步指南。我们专注于 dCas9 蛋白作为最先进的 DNA 靶向模块，与 10×SunTag 融合作为编辑增强最常用的系统。此外，我们讨论了为此目的使用的不同类型的 DNA 甲基转移酶模块，包括最近开发的最特异的变体。最后，我们解释了 gRNA 选择的原则，概述了细胞培养实验的设置，并简要介绍了可用的下游 DNA 甲基化数据分析选项。
