Journal
FRONTIERS IN PLANT SCIENCE
Volume 10, Issue -, Pages -Publisher
FRONTIERS MEDIA SA
DOI: 10.3389/fpls.2019.01150
Keywords
CRISPR/Cas9; genome editing; DsRED; Solanum lycopersicum; Oryza sativa; Arabidopsis thaliana
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Funding
- Spanish Ministry of Science, Innovation and Universities [AGL2014-57200-JIN, BFU2016-80621-P, BIO2016-78601-R]
- European Union H2020 program [634561, 679796, 760331-2, SEP-210417525]
- ERC grant SUMOrice
- BBSRC grant Flooding tolerance in rice
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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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