A modular toolbox for gRNA-Cas9 genome engineering in plants based on the GoldenBraid standard
[EN] Background: The efficiency, versatility and multiplexing capacity of RNA-guided genome engineering using the CRISPR/Cas9 technology enables a variety of applications in plants, ranging from gene editing to the construction of transcriptional gene circuits, many of which depend on the technical...
| Autores: | , , , , , , |
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| Tipo de recurso: | artículo |
| Fecha de publicación: | 2016 |
| País: | España |
| Institución: | Universitat Politècnica de València (UPV) |
| Repositorio: | RiuNet. Repositorio Institucional de la Universitat Politécnica de Valéncia |
| Idioma: | inglés |
| OAI Identifier: | oai:riunet.upv.es:10251/96423 |
| Acceso en línea: | https://riunet.upv.es/handle/10251/96423 |
| Access Level: | acceso abierto |
| Palabra clave: | Plant gene editing Plant gene activation Plant gene repression CRISPR/Cas9 gRNAs Multigenic assemblies GoldenBraid Luciferase/renilla assay GENETICA BIOQUIMICA Y BIOLOGIA MOLECULAR Aplicaciones de la biotecnología al diseño de nuevos carácteres y productos 32722 / W - Programa de doctorado en biotecnología 2071 Aplicaciones de la biotecnología al diseño de nuevos carácteres y productos 32722 / X - Máster universitario en biotecnología molecular y celular de plantas 2172 |
| Sumario: | [EN] Background: The efficiency, versatility and multiplexing capacity of RNA-guided genome engineering using the CRISPR/Cas9 technology enables a variety of applications in plants, ranging from gene editing to the construction of transcriptional gene circuits, many of which depend on the technical ability to compose and transfer complex synthetic instructions into the plant cell. The engineering principles of standardization and modularity applied to DNA cloning are impacting plant genetic engineering, by increasing multigene assembly efficiency and by fostering the exchange of well-defined physical DNA parts with precise functional information. Results: Here we describe the adaptation of the RNA-guided Cas9 system to GoldenBraid (GB), a modular DNA con¿ struction framework being increasingly used in Plant Synthetic Biology. In this work, the genetic elements required for CRISPRs-based editing and transcriptional regulation were adapted to GB, and a workflow for gRNAs construction was designed and optimized. New software tools specific for CRISPRs assembly were created and incorporated to the public GB resources site. Conclusions: The functionality and the efficiency of gRNA¿Cas9 GB tools were demonstrated in Nicotiana benthamiana using transient expression assays both for gene targeted mutations and for transcriptional regulation. The availability of gRNA¿Cas9 GB toolbox will facilitate the application of CRISPR/Cas9 technology to plant genome engineering |
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