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A modular toolbox for gRNA-Cas9 genome engineering in plants based on the GoldenBraid standard

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A modular toolbox for gRNA-Cas9 genome engineering in plants based on the GoldenBraid standard

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dc.contributor.author Vázquez-Vilar, Marta es_ES
dc.contributor.author Bernabé-Orts, Joan Miquel es_ES
dc.contributor.author Fernández Del Carmen, María Asunción es_ES
dc.contributor.author Ziarsolo Areitioaurtena, Pello es_ES
dc.contributor.author Blanca Postigo, José Miguel es_ES
dc.contributor.author Granell Richart, Antonio es_ES
dc.contributor.author Orzáez Calatayud, Diego Vicente es_ES
dc.date.accessioned 2018-02-02T10:07:04Z
dc.date.available 2018-02-02T10:07:04Z
dc.date.issued 2016 es_ES
dc.identifier.issn 1746-4811 es_ES
dc.identifier.uri http://hdl.handle.net/10251/96423
dc.description.abstract [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 es_ES
dc.description.sponsorship This work has been funded by Grant BIO2013-42193-R from Plan Nacional I + D of the Spanish Ministry of Economy and Competitiveness. Vazquez-Vilar M. is a recipient of a Junta de Ampliacion de Estudios fellowship. Bernabe-Orts J.M. is a recipient of a FPI fellowship. We want to thank Nicola J. Patron and Mark Youles for kindly providing humanCas9 and U6-26 clones. We also want to thank Eugenio Gomez for providing Arabidopsis thaliana genomic DNA and Concha Domingo for providing rice genomic DNA. We also want to thank the COST Action FA1006 for the support in the development of the software tools. es_ES
dc.language Inglés es_ES
dc.publisher Springer (Biomed Central Ltd.) es_ES
dc.relation MINECO/BIO2013-42193-R es_ES
dc.relation.ispartof Plant Methods es_ES
dc.rights Reconocimiento (by) es_ES
dc.subject Plant gene editing es_ES
dc.subject Plant gene activation es_ES
dc.subject Plant gene repression es_ES
dc.subject CRISPR/Cas9 es_ES
dc.subject gRNAs es_ES
dc.subject Multigenic assemblies es_ES
dc.subject GoldenBraid es_ES
dc.subject Luciferase/renilla assay es_ES
dc.subject.classification GENETICA es_ES
dc.subject.classification BIOQUIMICA Y BIOLOGIA MOLECULAR es_ES
dc.title A modular toolbox for gRNA-Cas9 genome engineering in plants based on the GoldenBraid standard es_ES
dc.type Artículo es_ES
dc.identifier.doi 10.1186/s13007-016-0101-2 es_ES
dc.rights.accessRights Abierto es_ES
dc.contributor.affiliation Universitat Politècnica de València. Instituto Universitario Mixto de Biología Molecular y Celular de Plantas - Institut Universitari Mixt de Biologia Molecular i Cel·lular de Plantes es_ES
dc.contributor.affiliation Universitat Politècnica de València. Departamento de Biotecnología - Departament de Biotecnologia es_ES
dc.contributor.affiliation Universitat Politècnica de València. Instituto Universitario de Conservación y Mejora de la Agrodiversidad Valenciana - Institut Universitari de Conservació i Millora de l'Agrodiversitat Valenciana es_ES
dc.description.bibliographicCitation Vázquez-Vilar, M.; Bernabé-Orts, JM.; Fernandez Del Carmen, MA.; Ziarsolo Areitioaurtena, P.; Blanca Postigo, JM.; Granell Richart, A.; Orzáez Calatayud, DV. (2016). A modular toolbox for gRNA-Cas9 genome engineering in plants based on the GoldenBraid standard. Plant Methods. 12. doi:10.1186/s13007-016-0101-2 es_ES
dc.description.accrualMethod S es_ES
dc.relation.publisherversion http://doi.org/10.1186/s13007-016-0101-2 es_ES
dc.type.version info:eu-repo/semantics/publishedVersion es_ES
dc.description.volume 12 es_ES
dc.identifier.pmid 26839579
dc.identifier.pmcid PMC4736081
dc.relation.pasarela S\306679 es_ES
dc.contributor.funder Ministerio de Economía y Competitividad es_ES
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