Targeted DNA methylation in human cells using engineered dCas9-methyltransferases
| Autor: | Marc Ostermeier, Fulya Turker, Winston Timp, Michael J. Spellberg, Glenna E. Meister, Rachael E. Workman, Tina Xiong, Carl D. Novina, Nathaniel C. Kato |
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| Jazyk: | angličtina |
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Models Molecular Protein Conformation alpha-Helical Methyltransferase Recombinant Fusion Proteins Science Genetic Vectors Computational biology Biology Protein Engineering Article Substrate Specificity 03 medical and health sciences Epigenetics of physical exercise CRISPR-Associated Protein 9 Escherichia coli Humans Protein Interaction Domains and Motifs DNA (Cytosine-5-)-Methyltransferases RNA-Directed DNA Methylation Epigenomics Genetics Gene Editing Multidisciplinary Binding Sites Base Sequence Methylation DNA Methylation Kinetics 030104 developmental biology HEK293 Cells CpG site DNA methylation Illumina Methylation Assay Medicine CpG Islands Protein Conformation beta-Strand Protein Binding RNA Guide Kinetoplastida |
| Zdroj: | Scientific Reports, Vol 7, Iss 1, Pp 1-14 (2017) Scientific Reports |
| ISSN: | 2045-2322 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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