Biochemical and Biophysical Research Communications, Vol.511, No.2, 246-252, 2019
Construction and validation of the CRISPR/dCas9-EZH2 system for targeted H3K27Me3 modification
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.