Institute for Transfusion Medicine and Gene Therapy, Medical Center-University of Freiburg, Freiburg, Germany.
Faculty of Biology, University of Freiburg, Freiburg, Germany.
Nat Protoc. 2020 Oct;15(10):3410-3440. doi: 10.1038/s41596-020-0380-y. Epub 2020 Sep 4.
The formation and function of highly specialized cells and tissues in a multicellular organism from a single genome are enabled through differential spatiotemporal access to the information contained in the genomic DNA. The epigenome plays an essential role in how DNA information can be accessed, and in the last decade the link between epigenetic aberrations and pathologies has become increasingly clear. Methods to precisely modify the epigenome are hence attracting interest as potential novel therapeutics. We recently described a platform, designer epigenome modifier (DEM), capable of precisely editing the epigenome of a cell to control the expression of selected genes. Here, we provide a detailed protocol to streamline the process of identifying DEMs that efficiently and selectively bind to their intended target site and inactivate expression of the target gene. Further, we describe the procedure to simultaneously regulate the expression of up to three genes in a multiplexed fashion. The protocol is divided into four stages that guide the user through the generation of the multicolor reporter cell line and its use for selecting functional DEMs. The duration of the whole procedure described varies from ~6 weeks when using a single reporter up to 13 weeks for fine-tuning the multiplex epigenome editing abilities of selected DEMs using three reporters. Given the great interest in epigenome editing in various fields of biomedical research, this protocol will help scientists to explore these novel technologies for their research.
多细胞生物中高度特化的细胞和组织是从单个基因组中形成和发育的，这一过程依赖于对基因组 DNA 中信息的差异时空访问。表观基因组在 DNA 信息如何被访问方面发挥着重要作用，在过去十年中，表观遗传异常与病理学之间的联系变得越来越清晰。因此，精确修饰表观基因组的方法作为潜在的新型治疗方法引起了人们的兴趣。我们最近描述了一种平台，即设计型表观基因组修饰剂（DEM），它能够精确地编辑细胞的表观基因组，以控制选定基因的表达。在这里，我们提供了一个详细的方案，以简化鉴定高效且选择性地与预期靶位点结合并使靶基因失活的 DEM 的过程。此外，我们还描述了以多路复用方式同时调节多达三个基因表达的程序。该方案分为四个阶段，指导用户生成多色报告细胞系，并用于选择功能型 DEM。整个过程的持续时间从使用单个报告基因时的大约 6 周到使用三个报告基因微调选定的 DEM 的多路复用表观基因组编辑能力时的 13 周不等。鉴于在生物医学研究的各个领域对表观基因组编辑的浓厚兴趣，本方案将帮助科学家探索这些新技术在其研究中的应用。
