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Targeted DNA methylation in human cells using engineered dCas9-methyltransferases

Xiong, Tina and Meister, Glenna E. and Workman, Rachael E. and Kato, Nathaniel C. and Spellberg, Michael J. and Turker, Fulya and Timp, Winston and Ostermeier, Marc and Novina, Carl D. (2017) Targeted DNA methylation in human cells using engineered dCas9-methyltransferases. Scientific Reports, 7 (1). ISSN 2045-2322

Abstract

Mammalian genomes exhibit complex patterns of gene expression regulated, in part, by DNA methylation. The advent of engineered DNA methyltransferases (MTases) to target DNA methylation to specific sites in the genome will accelerate many areas of biological research. However, targeted MTases require clear design rules to direct site-specific DNA methylation and minimize the unintended effects of off-target DNA methylation. Here we report a targeted MTase composed of an artificially split CpG MTase (sMTase) with one fragment fused to a catalytically-inactive Cas9 (dCas9) that directs the functional assembly of sMTase fragments at the targeted CpG site. We precisely map RNA-programmed DNA methylation to targeted CpG sites as a function of distance and orientation from the protospacer adjacent motif (PAM). Expression of the dCas9-sMTase in mammalian cells led to predictable and efficient (up to ~70%) DNA methylation at targeted sites. Multiplexing sgRNAs enabled targeting methylation to multiple sites in a single promoter and to multiple sites in multiple promoters. This programmable de novo MTase tool might be used for studying mechanisms of initiation, spreading and inheritance of DNA methylation, and for therapeutic gene silencing.

Item Type: Article
Date Deposited: 10 Aug 2017 00:45
Last Modified: 10 Aug 2017 00:45
URI: https://oak.novartis.com/id/eprint/33802

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