A CRISPR-based approach for targeted DNA demethylation.
A CRISPR-based approach for targeted DNA demethylation.
复制标题
基于 CRISPR 的靶向 DNA 去甲基化方法。
DOI：
10.1038/celldisc.2016.9
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发表时间：
2016
期刊：
影响因子：
33.5
通讯作者：
Hu R
中科院分区：
文献类型：
--
作者：
Xu X;Tao Y;Gao X;Zhang L;Li X;Zou W;Ruan K;Wang F;Xu GL;Hu R
在哺乳动物细胞中，DNA甲基化关键地调节基因表达，因此在无数生理和病理过程中具有关键作用。在这里，我们报告了一种新的靶向DNA去甲基化的方法，使用广泛使用的成簇规则间隔短回文重复序列（CRISPR）-Cas系统。最初，通过将两个拷贝的噬菌体MS 2 RNA元件插入到常规的sgRNA中来构建修饰的单向导RNA（sgRNA）（sgRNA2.0），这将促进Tetl催化结构域（Tet-CD）与dCas 9或MS 2外壳蛋白融合到靶基因座的拴系。随后，这种系统显示出显著上调靶基因的转录，包括RANKL、MAGEB 2或MMP 2，这与启动子中其相邻CpG的DNA去甲基化密切相关。此外，dCas 9/sgRNA2.0指导的去甲基化系统似乎提供了靶基因的有效去甲基化，具有微弱的脱靶效应。该系统的应用不仅可以帮助我们从机制上理解DNA甲基化如何在特定情况下调节基因表达，而且还可以控制基因表达和功能，具有潜在的临床益处。
In mammalian cells, DNA methylation critically regulates gene expression and thus has pivotal roles in myriad of physiological and pathological processes. Here we report a novel method for targeted DNA demethylation using the widely used clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system. Initially, modified single guide RNAs (sgRNAs) (sgRNA2.0) were constructed by inserting two copies of bacteriophage MS2 RNA elements into the conventional sgRNAs, which would facilitate the tethering of the Tet1 catalytic domain (Tet-CD), in fusion with dCas9 or MS2 coat proteins, to the targeted gene loci. Subsequently, such system was shown to significantly upregulate transcription of the target genes, including RANKL, MAGEB2 or MMP2, which was in close correlation to DNA demethylation of their neighboring CpGs in the promoters. In addition, the dCas9/sgRNA2.0-directed demethylation system appeared to afford efficient demethylation of the target genes with tenuous off-target effects. Applications of this system would not only help us understand mechanistically how DNA methylation might regulate gene expression in specific contexts, but also enable control of gene expression and functionality with potential clinical benefits.