The Jackson Laboratory for Genomic Medicine, 10 Discovery Drive, Farmington, CT, 06032, USA.
Center for Molecular Oncology, University of Connecticut Health, 263 Farmington Avenue, Farmington, CT, 06030, USA.
Nat Commun. 2019 Sep 20;10(1):4296. doi: 10.1038/s41467-019-12339-7.
Here we develop a methylation editing toolbox, Casilio-ME, that enables not only RNA-guided methylcytosine editing by targeting TET1 to genomic sites, but also by co-delivering TET1 and protein factors that couple methylcytosine oxidation to DNA repair activities, and/or promote TET1 to achieve enhanced activation of methylation-silenced genes. Delivery of TET1 activity by Casilio-ME1 robustly alters the CpG methylation landscape of promoter regions and activates methylation-silenced genes. We augment Casilio-ME1 to simultaneously deliver the TET1-catalytic domain and GADD45A (Casilio-ME2) or NEIL2 (Casilio-ME3) to streamline removal of oxidized cytosine intermediates to enhance activation of targeted genes. Using two-in-one effectors or modular effectors, Casilio-ME2 and Casilio-ME3 remarkably boost gene activation and methylcytosine demethylation of targeted loci. We expand the toolbox to enable a stable and expression-inducible system for broader application of the Casilio-ME platforms. This work establishes a platform for editing DNA methylation to enable research investigations interrogating DNA methylomes.
在这里，我们开发了一个甲基化编辑工具包 Casilio-ME，它不仅可以通过将 TET1 靶向基因组位点来实现 RNA 引导的甲基胞嘧啶编辑，还可以通过共递送 TET1 和将甲基胞嘧啶氧化偶联到 DNA 修复活性的蛋白因子，和/或促进 TET1 来实现对甲基化沉默基因的增强激活。Casilio-ME1 传递的 TET1 活性可显著改变启动子区域的 CpG 甲基化景观并激活甲基化沉默基因。我们对 Casilio-ME1 进行了增强，使其同时递送 TET1 催化结构域和 GADD45A（Casilio-ME2）或 NEIL2（Casilio-ME3），以简化氧化胞嘧啶中间产物的去除，从而增强靶向基因的激活。使用双效因子或模块化效应因子 Casilio-ME2 和 Casilio-ME3，可显著提高靶向基因座的基因激活和甲基胞嘧啶去甲基化。我们扩展了工具包，以实现 Casilio-ME 平台更广泛应用的稳定和表达诱导系统。这项工作建立了一个编辑 DNA 甲基化的平台，以支持对 DNA 甲基组学进行研究。
