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...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2017-07-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-017-06757-0 |
| _version_ | 1818840667419312128 |
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| 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 |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-12-19T04:13:50Z |
| format | Article |
| id | doaj.art-0f956c0b5d804d3494852eec91096851 |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-12-19T04:13:50Z |
| publishDate | 2017-07-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-0f956c0b5d804d3494852eec910968512022-12-21T20:36:20ZengNature PortfolioScientific Reports2045-23222017-07-017111410.1038/s41598-017-06757-0Targeted DNA methylation in human cells using engineered dCas9-methyltransferasesTina Xiong0Glenna E. Meister1Rachael E. Workman2Nathaniel C. Kato3Michael J. Spellberg4Fulya Turker5Winston Timp6Marc Ostermeier7Carl D. Novina8Department of Chemical and Biomolecular Engineering, Johns Hopkins UniversityDepartment of Cancer Immunology and Virology, Dana-Farber Cancer InstituteDepartment of Biomedical Engineering, Johns Hopkins UniversityDepartment of Chemical and Biomolecular Engineering, Johns Hopkins UniversityDepartment of Cancer Immunology and Virology, Dana-Farber Cancer InstituteDepartment of Cancer Immunology and Virology, Dana-Farber Cancer InstituteDepartment of Biomedical Engineering, Johns Hopkins UniversityDepartment of Chemical and Biomolecular Engineering, Johns Hopkins UniversityDepartment of Cancer Immunology and Virology, Dana-Farber Cancer InstituteAbstract 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.https://doi.org/10.1038/s41598-017-06757-0 |
| spellingShingle | 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 Scientific Reports |
| title | 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_short | Targeted DNA methylation in human cells using engineered dCas9-methyltransferases |
| title_sort | targeted dna methylation in human cells using engineered dcas9 methyltransferases |
| url | https://doi.org/10.1038/s41598-017-06757-0 |
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