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Identification of Transgene-Free CRISPR-Edited Plants of Rice, Tomato, and Arabidopsis by Monitoring DsRED Fluorescence in Dry Seeds

Aliaga-Franco, Norma; Zhang, Cunjin; Presa, Silvia; Srivastava, Anjil K.; Granell, Antonio; Alabadí, David; Sadanandom, Ari; Blázquez, Miguel A.; Minguet, Eugenio G.

Identification of Transgene-Free CRISPR-Edited Plants of Rice, Tomato, and Arabidopsis by Monitoring DsRED Fluorescence in Dry Seeds Thumbnail


Authors

Norma Aliaga-Franco

Silvia Presa

Anjil K. Srivastava

Antonio Granell

David Alabadí

Miguel A. Blázquez

Eugenio G. Minguet



Abstract

Efficient elimination of the editing machinery remains a challenge in plant biotechnology after genome editing to minimize the probability of off-target mutations, but it is also important to deliver end users with edited plants free of foreign DNA. Using the modular cloning system Golden Braid, we have included a fluorescence-dependent transgene monitoring module to the genome-editing tool box. We have tested this approach in Solanum lycopersicum, Oryza sativa, and Arabidopsis thaliana. We demonstrate that DsRED fluorescence visualization works efficiently in dry seeds as marker for the detection of the transgene in the three species allowing an efficient method for selecting transgene-free dry seeds. In the first generation of DsRED-free CRISPR/Cas9 null segregants, we detected gene editing of selected targets including homozygous mutants for the plant species tested. We demonstrate that this strategy allows rapid selection of transgene-free homozygous edited crop plants in a single generation after in vitro transformation.

Citation

Aliaga-Franco, N., Zhang, C., Presa, S., Srivastava, A. K., Granell, A., Alabadí, D., Sadanandom, A., Blázquez, M. A., & Minguet, E. G. (2019). Identification of Transgene-Free CRISPR-Edited Plants of Rice, Tomato, and Arabidopsis by Monitoring DsRED Fluorescence in Dry Seeds. Frontiers in Plant Science, 10, Article 1150. https://doi.org/10.3389/fpls.2019.01150

Journal Article Type Article
Acceptance Date Aug 23, 2019
Online Publication Date Sep 18, 2019
Publication Date Sep 18, 2019
Deposit Date Oct 8, 2019
Publicly Available Date Oct 8, 2019
Journal Frontiers in Plant Science
Publisher Frontiers Media
Peer Reviewed Peer Reviewed
Volume 10
Article Number 1150
DOI https://doi.org/10.3389/fpls.2019.01150
Public URL https://durham-repository.worktribe.com/output/1318722

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Publisher Licence URL
http://creativecommons.org/licenses/by/4.0/

Copyright Statement
© 2019 Aliaga-Franco, Zhang, Presa, Srivastava, Granell, Alabadí,
Sadanandom, Blázquez and Minguet. This is an open-access article distributed
under the terms of the Creative Commons Attribution License (CC BY). The use,
distribution or reproduction in other forums is permitted, provided the original
author(s) and the copyright owner(s) are credited and that the original publication
in this journal is cited, in accordance with accepted academic practice. No use,
distribution or reproduction is permitted which does not comply with these terms.






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