New cloning system allows development of transgene-free edited crops

Researchers from Polytechnic University of Valencia, Spain and Durham University in the UK developed a tried and tested cloning system for genome editing with a monitoring module that lights up when a transgene is detected. This new technique provides an option for researchers to have an efficient elimination of editing system after genome editing, minimizing the chance of off-target mutations, and also allows development of edited plants free of foreign DNA.

 

The new modular cloning system called Golden Braid has been tested in tomatoes, rice, and Arabidopsis. It was found that the fluorescence visualization worked well in dry seeds, indicating the presence of transgenes in tomato, rice, and Arabidopsis, which allows an efficient way to select transgene-free T2 dry seeds. The researchers detected gene editing of selected traits in T2 plants and identified homozygous gene-edited plants with different mutations.

 

Results of the study published in Frontiers in Plant Science show that the developed strategy allows fast collection of transgene-free edited crop plants after just one generation after transformation.

Article:
Identification of transgene-free CRISPR edited plants of rice, tomato and Arabidopsis by monitoring DsRED fluorescence in dry seeds, Frontiers in Plant Science, doi: 10.3389/fpls.2019.01150

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 T2 dry seeds. We detected gene editing of selected targets in T2 plants, but more importantly, we identified homozygous gene edited plants with different mutations in 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.