Construction and validation of the CRISPR/dCas9-EZH2 system for targeted H3K27Me3 modification
Construction and validation of the CRISPR/dCas9-EZH2 system for targeted H3K27Me3 modification
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用于靶向 H3K27Me3 修饰的 CRISPR/dCas9-EZH2 系统的构建和验证
DOI：
10.1016/j.bbrc.2019.02.011
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发表时间：
2019-04-02
影响因子：
3.1
通讯作者：
Song, Yingliang
中科院分区：
文献类型：
--
作者：
Chen, Xutao;Wei, Mengying;Song, Yingliang
细胞表型与表观基因组密切相关，可以通过有针对性地操纵表观遗传标记来精确调节。在这里，我们成功地构建了一个靶向组蛋白甲基化系统，该系统由无核酸酶的dCas9蛋白、与PP7 RNA适配体融合的sgRNA和与PP7外壳蛋白融合的Zeste Homolog 2增强子（EZH2）组成。在dCas9/sgRNA-PP7的引导下，PCP-EZH2可以特异性靶向基因位点，催化组蛋白H3赖氨酸27的3甲基化，从而抑制基因表达。这种基因抑制系统被认为是高效、特异和灵活的。作为一项概念验证研究，我们设计了靶向C/ebp α启动子区域的sgRNA。在dCas9、sgRNA/C/ebp α - pp7和PCP-EZH2共感染的细胞中，通过诱导C/ebp α启动子上H3K27的三甲基化，C/ebp α基因的表达显著降低，其结果在子细胞中具有表观遗传性。总之，我们的研究结果成功建立了由dCas9/sgRNA-PP7和PCP-EZH2组成的基因修饰体系，为基因表观遗传修饰和表达的靶向操作提供了强有力的工具。(C) 2019由爱思唯尔公司出版。
Cell phenotypes are closely related to the epigenome, which could be precisely regulated by the targeted manipulation of epigenetic marks. Here, we have successfully produced a targeted histone methylation system, which consists of nuclease-null dCas9 protein, the sgRNA fused with PP7 RNA aptamers and the Enhancer of Zeste Homolog 2 (EZH2) fused to PP7 coat protein (PCP). Guided by the dCas9/sgRNA-PP7, the PCP-EZH2 can specifically target gene loci to catalyze 3 methylation of histone H3 lysine 27, resulting in the inhibition of gene expression. This kind of gene inhibition system is supposed to be highly effective, specific and flexible. As a proof-of-concept study, sgRNA targeting C/ebp alpha promoter region was designed. In the cells co-infected with the dCas9, sgRNA/C/ebp alpha-PP7 and PCP-EZH2, the expression of C/ebp alpha gene was significantly reduced via induction of trimethylation to H3K27 on C/ebp alpha promoter, with the results epigenetically inherited in the daughter cells. In conclusion, our results successfully established a gene modification system consisting of dCas9/sgRNA-PP7 and PCP-EZH2, providing a robust tool for targeted manipulation of gene epigenetic modification and expression. (C) 2019 Published by Elsevier Inc.