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Cell Discov. 2016 May 3;2:16009. doi: 10.1038/celldisc.2016.9. eCollection 2016.

A CRISPR-based approach for targeted DNA demethylation.

Author information

1
Key Laboratory of Systems Biology, CAS Center for Excellence in Molecular Cell Science, Innovation Center for Cell Signaling Network; Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences , Shanghai, China.
2
Key Laboratory of Systems Biology, CAS Center for Excellence in Molecular Cell Science, Innovation Center for Cell Signaling Network; Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China; University of Chinese Academy of Sciences, Shanghai, China.
3
State Key Laboratory of Cell Biology , Shanghai, China.
4
State Key Laboratory of Molecular Biology; Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences , Shanghai, China.
5
Departments of Oral and Craniofacial Implant and Oral and Maxillofacial Surgery, School of Medicine, the Ninth People's Hospital Affiliated with Shanghai Jiao Tong University , Shanghai, China.

Abstract

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.

KEYWORDS:

CRISPR; RANKL; demethylation

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