Enhancing tomato fruit sweetness by CRISPR/Cas9-mediated SlVIF gene editing

In tomato plants, vacuolar invertase inhibitor (VIF) is a negative regulator of sucrose (SUC) degradation into fructose (FRU) and glucose (GLU), suggesting knockout of SlVIF promotes the accumulation of FRU and GLU. A dual-target vector of pKSE402- SlVIF was constructed and introduced into the tomato breeding parent ‘1912’ via Agrobacterium -mediated transformation. The editing efficiency was as high as 47%. Two Cas9-free T 1 lines exhibited delayed fruit ripening, significantly increased FRU and GLU levels, and reduced SUC content at the red ripe stage compared to wild-type (WT) plants, while fruit size and yield were unchanged. Additionally, in the SlVIF knockout mutants, the VIF protein structure was significantly altered, and VIN enzyme activity was markedly increased, which may underlie the enhanced degradation of sucrose in tomato fruits. The findings indicate that the sweetness of tomato fruit can be improved by CRISPR/Cas9-mediated SlVIF gene editing, which provides valuable resources for high-quality breeding in tomato. • VIF is a regulator of sucrose metabolism, suggesting a key role in fruit sweetness. • Knockout of SlVIF increases the activity of VIN enzyme . • Knockout of SlVIF increases fructose and glucose contents, and decreases sucrose content. • Knockout of SlVIF delays fruit ripening and extends shelf life.