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Utilizing RNA origami scaffolds in Saccharomyces cerevisiae for dCas9-mediated transcriptional control
Designer RNA scaffolds constitute a promising tool for synthetic biology, as they can be genetically expressed to perform specific functions in vivo such as scaffolding enzymatic cascades and regulating gene expression through CRISPR-dCas9 applications. RNA origami is a recently developed RNA design...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9262615/ https://www.ncbi.nlm.nih.gov/pubmed/35648481 http://dx.doi.org/10.1093/nar/gkac470 |
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author | Pothoulakis, Georgios Nguyen, Michael T A Andersen, Ebbe S |
author_facet | Pothoulakis, Georgios Nguyen, Michael T A Andersen, Ebbe S |
author_sort | Pothoulakis, Georgios |
collection | PubMed |
description | Designer RNA scaffolds constitute a promising tool for synthetic biology, as they can be genetically expressed to perform specific functions in vivo such as scaffolding enzymatic cascades and regulating gene expression through CRISPR-dCas9 applications. RNA origami is a recently developed RNA design approach that allows construction of large RNA nanostructures that can position aptamer motifs to spatially organize other molecules, including proteins. However, it is still not fully understood how positioning multiple aptamers on a scaffold and the orientation of a scaffold affects functional properties. Here, we investigate fusions of single-guide RNAs and RNA origami scaffolds (termed sgRNAO) capable of recruiting activating domains for control of gene expression in yeast. Using MS2 and PP7 as orthogonal protein-binding aptamers, we observe a gradual increase in transcriptional activation for up to four aptamers. We demonstrate that different aptamer positions on a scaffold and scaffold orientation affect transcriptional activation. Finally, sgRNAOs are used to regulate expression of enzymes of the violacein biosynthesis pathway to control metabolic flux. The integration of RNA origami nanostructures at promoter sites achieved here, can in the future be expanded by the addition of functional motifs such as riboswitches, ribozymes and sensor elements to allow for complex gene regulation. |
format | Online Article Text |
id | pubmed-9262615 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-92626152022-07-08 Utilizing RNA origami scaffolds in Saccharomyces cerevisiae for dCas9-mediated transcriptional control Pothoulakis, Georgios Nguyen, Michael T A Andersen, Ebbe S Nucleic Acids Res Synthetic Biology and Bioengineering Designer RNA scaffolds constitute a promising tool for synthetic biology, as they can be genetically expressed to perform specific functions in vivo such as scaffolding enzymatic cascades and regulating gene expression through CRISPR-dCas9 applications. RNA origami is a recently developed RNA design approach that allows construction of large RNA nanostructures that can position aptamer motifs to spatially organize other molecules, including proteins. However, it is still not fully understood how positioning multiple aptamers on a scaffold and the orientation of a scaffold affects functional properties. Here, we investigate fusions of single-guide RNAs and RNA origami scaffolds (termed sgRNAO) capable of recruiting activating domains for control of gene expression in yeast. Using MS2 and PP7 as orthogonal protein-binding aptamers, we observe a gradual increase in transcriptional activation for up to four aptamers. We demonstrate that different aptamer positions on a scaffold and scaffold orientation affect transcriptional activation. Finally, sgRNAOs are used to regulate expression of enzymes of the violacein biosynthesis pathway to control metabolic flux. The integration of RNA origami nanostructures at promoter sites achieved here, can in the future be expanded by the addition of functional motifs such as riboswitches, ribozymes and sensor elements to allow for complex gene regulation. Oxford University Press 2022-06-01 /pmc/articles/PMC9262615/ /pubmed/35648481 http://dx.doi.org/10.1093/nar/gkac470 Text en © The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research. https://creativecommons.org/licenses/by/4.0/This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Synthetic Biology and Bioengineering Pothoulakis, Georgios Nguyen, Michael T A Andersen, Ebbe S Utilizing RNA origami scaffolds in Saccharomyces cerevisiae for dCas9-mediated transcriptional control |
title | Utilizing RNA origami scaffolds in Saccharomyces cerevisiae for dCas9-mediated transcriptional control |
title_full | Utilizing RNA origami scaffolds in Saccharomyces cerevisiae for dCas9-mediated transcriptional control |
title_fullStr | Utilizing RNA origami scaffolds in Saccharomyces cerevisiae for dCas9-mediated transcriptional control |
title_full_unstemmed | Utilizing RNA origami scaffolds in Saccharomyces cerevisiae for dCas9-mediated transcriptional control |
title_short | Utilizing RNA origami scaffolds in Saccharomyces cerevisiae for dCas9-mediated transcriptional control |
title_sort | utilizing rna origami scaffolds in saccharomyces cerevisiae for dcas9-mediated transcriptional control |
topic | Synthetic Biology and Bioengineering |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9262615/ https://www.ncbi.nlm.nih.gov/pubmed/35648481 http://dx.doi.org/10.1093/nar/gkac470 |
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