Efficient removal of the editing machinery continues to be a concern in genome editing. To address this challenge, researchers at Universidad Politécnica de Valencia in Spain and Durham University in the UK used the modular cloning system Golden Braid and included a fluorescence-dependent transgene monitoring module to the genome-editing toolbox.

The technique was tested in tomato, rice, and Arabidopsis which showed that DsRED fluorescence visualization worked well in dry seeds as marker for the detection of the transgene. This shows that the method is an efficient way to select transgene-free dry seeds. In the first generation of Ds-RED-free CRISPR-Cas9 null segregants, gene editing of selected targets such as homozygous mutants was detected.

The results of the study indicate that the technique allows fast selection of transgene-free homozygous edited crop plants in a single generation after in vitro transformation.

Read the research article in Frontiers in Plant Science.

Figure 1 (A) Position of each CRISPR target in selected genes At5g55250 (Arabidopsis thaliana), Solyc07g64990 and Solyc12g14500 (Solanum lycopersicon), and Os04g56950 (Oryza sativa). (B) Description of transcriptional units (arrows) assembled in the vectors generated for plant transformation using the modular system GoldenBraid. Specific promoters were selected for expression of Cas9 protein in each species, pAtUBQ10 in Arabidopsis thaliana, p35S for Solanum Lycopersicum, and pZmUBQ for Oryza sativa. Adequate promoter for expression of sgRNA was selected: pAtU6-26 for dicotyledonous species (Solanum and Arabidopsis) and pOsU3 for monocotyledonous species (rice).