CRISPR/Cas9-Mediated Mutagenesis in Tomato Targeting the DE-ETIOLATED1 Gene

Tomato high pigment-2 (hp-2dg, hp-2, and hp-2j) mutant lines are characterized by mutations in the DE-ETIOLATED1 (SlDET1; Solyc01g056340) gene. SlDET1 is responsible for encoding a nuclear protein that acts as a negative regulator involved in light signaling, repressing photomorphogenesis. These tomato mutant lines are known for increased levels of antioxidant pigments in fruits, such as flavonoids and carotenoids, compared to the wild-type fruits. In this study, CRISPR/Cas9, followed by the non-homologous end joining mechanism of repair (NHEJ), was used to target the SlDET1 gene and investigate whether the effects of these mutations could mimic the effects of hp-2 mutant lines, improving the nutritional features of tomato fruits. Our results indicated that mutations generated by CRISPR/Cas9 NHEJ in the hp-2 and hp-2j regions (exon 11) resulted in significant changes in the SlDET1 coding and protein sequences. These mutations caused a low survival rate of edited sprouts and regenerated plants with a very compromised capacity of allelic heritability of these mutations for the following generations. However, regenerated plants containing these site-specific mutations in the SlDET1 gene showed higher levels of phytochemicals in ripe fruits. Furthermore, these edited plants also showed an upregulation of structural genes involved in the synthesis of these biocompounds. Although the SlDET1 gene could be considered an interesting target gene for the nutritional improvement of tomato fruits, our results showed that mutations within its exon 11 are quite critical and can induce severe perturbations in plant physiology, with a compromised possibility to develop new stable edited lines.