Targeted DNA methylation in human cells using engineered dCas9-methyltransferases

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 MTase...

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Autores principales:	Tina Xiong, Glenna E. Meister, Rachael E. Workman, Nathaniel C. Kato, Michael J. Spellberg, Fulya Turker, Winston Timp, Marc Ostermeier, Carl D. Novina
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/0f956c0b5d804d3494852eec91096851
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spelling	oai:doaj.org-article:0f956c0b5d804d3494852eec910968512021-12-02T15:06:13ZTargeted DNA methylation in human cells using engineered dCas9-methyltransferases10.1038/s41598-017-06757-02045-2322https://doaj.org/article/0f956c0b5d804d3494852eec910968512017-07-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-06757-0https://doaj.org/toc/2045-2322Abstract 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.Tina XiongGlenna E. MeisterRachael E. WorkmanNathaniel C. KatoMichael J. SpellbergFulya TurkerWinston TimpMarc OstermeierCarl D. NovinaNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-14 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Tina Xiong Glenna E. Meister Rachael E. Workman Nathaniel C. Kato Michael J. Spellberg Fulya Turker Winston Timp Marc Ostermeier Carl D. Novina Targeted DNA methylation in human cells using engineered dCas9-methyltransferases
description	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.
format	article
author	Tina Xiong Glenna E. Meister Rachael E. Workman Nathaniel C. Kato Michael J. Spellberg Fulya Turker Winston Timp Marc Ostermeier Carl D. Novina
author_facet	Tina Xiong Glenna E. Meister Rachael E. Workman Nathaniel C. Kato Michael J. Spellberg Fulya Turker Winston Timp Marc Ostermeier Carl D. Novina
author_sort	Tina Xiong
title	Targeted DNA methylation in human cells using engineered dCas9-methyltransferases
title_short	Targeted DNA methylation in human cells using engineered dCas9-methyltransferases
title_full	Targeted DNA methylation in human cells using engineered dCas9-methyltransferases
title_fullStr	Targeted DNA methylation in human cells using engineered dCas9-methyltransferases
title_full_unstemmed	Targeted DNA methylation in human cells using engineered dCas9-methyltransferases
title_sort	targeted dna methylation in human cells using engineered dcas9-methyltransferases
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/0f956c0b5d804d3494852eec91096851
work_keys_str_mv	AT tinaxiong targeteddnamethylationinhumancellsusingengineereddcas9methyltransferases AT glennaemeister targeteddnamethylationinhumancellsusingengineereddcas9methyltransferases AT rachaeleworkman targeteddnamethylationinhumancellsusingengineereddcas9methyltransferases AT nathanielckato targeteddnamethylationinhumancellsusingengineereddcas9methyltransferases AT michaeljspellberg targeteddnamethylationinhumancellsusingengineereddcas9methyltransferases AT fulyaturker targeteddnamethylationinhumancellsusingengineereddcas9methyltransferases AT winstontimp targeteddnamethylationinhumancellsusingengineereddcas9methyltransferases AT marcostermeier targeteddnamethylationinhumancellsusingengineereddcas9methyltransferases AT carldnovina targeteddnamethylationinhumancellsusingengineereddcas9methyltransferases
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Targeted DNA methylation in human cells using engineered dCas9-methyltransferases

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