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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 and Zhang, Cunjin and Presa, Silvia and Srivastava, Anjil K. and Granell, Antonio and Alabadí, David and Sadanandom, Ari and Blázquez, Miguel A. and Minguet, Eugenio 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 . p. 1150.


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.

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Publisher 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.
Date accepted:23 August 2019
Date deposited:08 October 2019
Date of first online publication:18 September 2019
Date first made open access:08 October 2019

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