Efficient targeted DNA methylation with chimeric dCas9-Dnmt3a-Dnmt3L methyltransferase.
Efficient targeted DNA methylation with chimeric dCas9-Dnmt3a-Dnmt3L methyltransferase.
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DOI：
10.1093/nar/gkw1112
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发表时间：
2017-02-28
影响因子：
14.9
通讯作者：
Jurkowski TP
中科院分区：
文献类型：
--
作者：
Stepper P;Kungulovski G;Jurkowska RZ;Chandra T;Krueger F;Reinhardt R;Reik W;Jeltsch A;Jurkowski TP
DNA甲基化在调节和维持细胞类型特异性转录程序中起着关键作用。靶向表观基因组编辑是一种新兴技术，用于特异性调节细胞基因表达，以调节细胞表型或剖析参与其控制的表观遗传机制。在这项工作中，我们采用了与核酸酶失活的dCas 9可编程靶向结构域融合的DNA甲基转移酶Dnmt 3a-Dnmt 3L构建体，以将DNA甲基化引入人类基因组中，特异性地在EpCAM、CXCR 4和TFRC基因启动子处。我们表明，用单个gRNA靶向这些基因座导致启动子的有效和广泛的甲基化。几种指导RNA的多重化不会增加甲基化的效率。在PAM位点的上游25 bp和下游40 bp附近观察到靶向甲基化的峰，而结合位点本身的20-30 bp被保护免于甲基化。有效的甲基化依赖于DNA上Dnmt 3a/Dnmt 3L复合物的多聚化。此外，引入的甲基化导致靶基因的转录抑制。这些新的可编程表观遗传编辑器允许对细胞中DNA甲基化状态进行前所未有的控制，并将导致对表观遗传信号的理解的进一步进展。
DNA methylation plays a critical role in the regulation and maintenance of cell-type specific transcriptional programs. Targeted epigenome editing is an emerging technology to specifically regulate cellular gene expression in order to modulate cell phenotypes or dissect the epigenetic mechanisms involved in their control. In this work, we employed a DNA methyltransferase Dnmt3a–Dnmt3L construct fused to the nuclease-inactivated dCas9 programmable targeting domain to introduce DNA methylation into the human genome specifically at the EpCAM, CXCR4 and TFRC gene promoters. We show that targeting of these loci with single gRNAs leads to efficient and widespread methylation of the promoters. Multiplexing of several guide RNAs does not increase the efficiency of methylation. Peaks of targeted methylation were observed around 25 bp upstream and 40 bp downstream of the PAM site, while 20–30 bp of the binding site itself are protected against methylation. Potent methylation is dependent on the multimerization of Dnmt3a/Dnmt3L complexes on the DNA. Furthermore, the introduced methylation causes transcriptional repression of the targeted genes. These new programmable epigenetic editors allow unprecedented control of the DNA methylation status in cells and will lead to further advances in the understanding of epigenetic signaling.