Comparative transcriptional responses to two CRISPR/Cas9 target constructs targeting the tomato SlLCYB-2 Gene using a transient expression system

The CRISPR/Cas9 system has become a powerful tool for plant genome editing.However, even within the same gene, the degree of gene suppression may vary depending on the selected target site, and the factors influencing target site performance remain insufficiently understood.In this study, two different CRISPR/Cas9 target sites (Target1 and Target2) were designed within the lycopene β-cyclase-2 (SlLCYB-2) gene of tomato (Solanum lycopersicum), and their effects on gene expression were compared using an Agrobacterium-mediated transient expression system.Reduced SlLCYB-2 transcript levels were observed following treatment with both target constructs, with a greater reduction observed in the Target 2 treatment than in the Target 1 treatment.The reduced SlLCYB-2 transcript levels were accompanied by target-dependent transcriptional responses of carotenoid metabolism-related genes, including β-carotene hydroxylase 1, β-carotene hydroxylase 2, and zeaxanthin epoxidase.These findings indicate that the selection of CRISPR/Cas9 target sites influences the extent of SlLCYB-2 transcriptional suppression and the transcriptional responses of downstream carotenoid metabolism-related genes.Collectively, our results demonstrate that target site selection is an important factor influencing CRISPR/Cas9-mediated transcriptional regulation in tomato.