Biosignal Genome Resource Center, IMCR, Gunma University, Maebashi, Japan.
Methods Mol Biol. 2024;2842:155-165. doi: 10.1007/978-1-0716-4051-7_7.
DNA methylation, one of the most studied epigenetic modifications, regulates many biological processes. Dysregulation of DNA methylation is implicated in the etiology of several diseases, such as cancer and imprinting diseases. Accordingly, technologies designed to manipulate DNA methylation at specific loci are considered worthwhile and many epigenome editing technologies have been developed, which were based on ZF, TALE, and CRISPR-dCas9. Here, we describe a protocol for the application of a modified dCas9-SunTag system, which increased the efficiency of targeted demethylation and gene activation at specific DNA loci. The original SunTag system consists of 10 copies of the GCN4 peptide separated by 5-amino-acid linkers. To achieve more efficient recruitment of an anti-GCN4 scFv fused to the ten-eleven (TET) 1 hydroxylase, an enzyme that demethylates DNA, we changed the linker length to 22 amino acids. Moreover, we describe the co-recruitment of TET1 and VP64 for efficient gene activation. Since we showed the manipulation of DNA methylation at specific loci and gene activation, its application could lead to its future use in the clinic.
DNA 甲基化是研究最多的表观遗传修饰之一，它调节着许多生物过程。DNA 甲基化的失调与许多疾病的发病机制有关，如癌症和印记疾病。因此，设计用于在特定基因座上操纵 DNA 甲基化的技术被认为是有价值的，并且已经开发了许多表观基因组编辑技术，这些技术基于 ZF、TALE 和 CRISPR-dCas9。在这里，我们描述了一种应用改良的 dCas9-SunTag 系统的方案，该系统提高了在特定 DNA 基因座上靶向去甲基化和基因激活的效率。原始的 SunTag 系统由 10 个 GCN4 肽组成，由 5 个氨基酸接头隔开。为了更有效地募集与十-十一（TET）1 羟化酶融合的抗 GCN4 scFv，该酶可使 DNA 去甲基化，我们将接头长度更改为 22 个氨基酸。此外，我们还描述了 TET1 和 VP64 的共同募集，以实现有效的基因激活。由于我们展示了在特定基因座上操纵 DNA 甲基化和基因激活，因此其应用可能会导致其未来在临床上的应用。
