The GB4.0 Platform, an All-In-One Tool for CRISPR/Cas-Based Multiplex Genome Engineering in Plants
| Autor: | Antonio Granell, Marta Vazquez-Vilar, Asun Fernández-del-Carmen, José Carlos Quintela, Maria Ajenjo, Diego Orzaez, Arianna Ressa, Victor Garcia-Carpintero, Javier Sanchez-Vicente, Carmine de Paola, Joan Miquel Bernabé-Orts, Sara Selma, Blanca Salazar-Sarasua |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
Genome engineering
0106 biological sciences 0301 basic medicine Nicotiana benthamiana Computational biology Plant Science 01 natural sciences Genome tobacco Multiplexing SB1-1110 03 medical and health sciences CRISPR/Cas Tobacco BIOQUIMICA Y BIOLOGIA MOLECULAR CRISPR Gene family Gene Original Research Trans-activating crRNA multiplexing biology Cas9 fungi Plant culture food and beverages GoldenBraid biology.organism_classification 030104 developmental biology CRISPR Cas genome engineering 010606 plant biology & botany |
| Zdroj: | Frontiers in Plant Science Frontiers in Plant Science, Vol 12 (2021) RiuNet. Repositorio Institucional de la Universitat Politécnica de Valéncia instname Digital.CSIC. Repositorio Institucional del CSIC |
| ISSN: | 1664-462X |
| Popis: | [EN] CRISPR/Cas ability to target several loci simultaneously (multiplexing) is a game-changer in plant breeding. Multiplexing not only accelerates trait pyramiding but also can unveil traits hidden by functional redundancy. Furthermore, multiplexing enhances dCas-based programmable gene expression and enables cascade-like gene regulation. However, the design and assembly of multiplex constructs comprising tandemly arrayed guide RNAs (gRNAs) requires scarless cloning and is still troublesome due to the presence of repetitive sequences, thus hampering a more widespread use. Here we present a comprehensive extension of the software-assisted cloning platform GoldenBraid (GB), in which, on top of its multigene cloning software, we integrate new tools for the Type IIS-based easy and rapid assembly of up to six tandemly-arrayed gRNAs with both Cas9 and Cas12a, using the gRNA-tRNA-spaced and the crRNA unspaced approaches, respectively. As stress tests for the new tools, we assembled and used for Agrobacterium-mediated stable transformation a 17 Cas9-gRNAs construct targeting a subset of the Squamosa-Promoter Binding Protein-Like (SPL) gene family in Nicotiana tabacum. The 14 selected genes are targets of miR156, thus potentially playing an important role in juvenile-to-adult and vegetative-to-reproductive phase transitions. With the 17 gRNAs construct we generated a collection of Cas9-free SPL edited T-1 plants harboring up to 9 biallelic mutations and showing leaf juvenility and more branching. The functionality of GB-assembled dCas9 and dCas12a-based CRISPR/Cas activators and repressors using single and multiplexing gRNAs was validated using a Luciferase reporter with the Solanum lycopersicum Mtb promoter or the Agrobacterium tumefaciens nopaline synthase promoter in transient expression in Nicotiana benthamiana. With the incorporation of the new web-based tools and the accompanying collection of DNA parts, the GB4.0 genome edition turns an all-in-one open platform for plant genome engineering. This work had been funded by EU Horizon 2020 Project Newcotiana Grant 760331 and PID2019-108203RB-100 Plan Nacional I+D, Spanish Ministry of Economy and Competitiveness. MV-V was recipient of aGeneralitat Valenciana and Fondo Social Europeo post-doctoral grant. JB-O and SS were recipients of FPI fellowships. CP was recipient of a Santiago Grisolia fellowship (Generalitat Valenciana). |
| Databáze: | OpenAIRE |
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