Fine-tuning RIPENING INHIBITOR ( RIN) expression by introducing allelic mutations in its promoter using CRISPR/Cas9 multiplex editing

Tomato is one of the most produced and consumed vegetables globally due to its nutritional benefits, sensory characteristics, and cultural importance. However, tomato fruit has a short shelf-life, which can be extended by postharvest techniques, but often at the expense of fruit quality, leading to consumer dissatisfaction. To address this challenge, we modified the upstream regulatory regions of Ripening inhibitor (RIN), a master regulator of tomato fruit ripening, utilizing a CRISPR/Cas9 multiplex system. This approach enabled the creation of a population of tomato fruit with mutations of varying severity, which could have far-reaching effects on the RIN-induced gene regulatory network in tomato fruit, leading to downstream changes in fruit traits. We have generated 264 first-generation (T ₀ ) transgenic lines of RIN promoter mutants with diverse genetic lesions and RIN transcriptional levels. Our study revealed a non-linear relationship between promoter mutations and gene expression, highlighting the potential roles of certain types of mutations in regulating RIN transcription. Future work will focus on evaluating fruit traits from mutants with pronounced changes in RIN expression, as well as performing transcriptomic analysis to explore the mechanisms underlying fruit quality modifications due to genome editing.