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Nucleic Acids Res. 2017 Feb 28;45(4):1703-1713. doi: 10.1093/nar/gkw1112.

Efficient targeted DNA methylation with chimeric dCas9-Dnmt3a-Dnmt3L methyltransferase.

Author information

1
Institute of Biochemistry, Pfaffenwaldring 55, Faculty of Chemistry, University of Stuttgart, D-70569 Stuttgart, Germany.
2
Epigenetics Programme, The Babraham Institute, Cambridge CB22 3AT, UK; The Welcome Trust Sanger Institute, Cambridge CB10 1SA, UK.
3
The Babraham Institute, Cambridge CB22 3AT, UK.
4
Max Planck Genome-Centre Cologne, Carl-von-Linné-Weg 10, D-50829 Cologne, Germany.

Abstract

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.

PMID:
27899645
PMCID:
PMC5389507
DOI:
10.1093/nar/gkw1112
[Indexed for MEDLINE]
Free PMC Article

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