This study investigates the regulatory roles of transcription factors (TFs) in tomato fruit ripening and metabolite accumulation. Using CRISPR/Cas9-mediated mutagenesis, the authors generated genetically engineered tomato lines with altered TF expression and analyzed their effects on fruit metabolites and transcript profiles. Key findings include: 1. **TFs Regulate Carotenoid Content**: Mutations in specific TFs, such as *HY5*, *AP2a*, and *TAGL1*, significantly altered carotenoid contents in tomato fruits. For example, *HY5* knockout increased carotenoid content by 30%, while *AP2a* knockout decreased it by 38%. 2. **TFs Influence Sugar Accumulation**: The *HY5* mutant showed a 20% increase in fructose and glucose content compared to wild-type (WT) fruits. Further analysis revealed that *HY5* directly binds to the *SWEET12c* promoter, activating its expression and modulating sugar transport. 3. **TFs Modulate ABA and Phenolic Acid Biosynthesis**: Mutations in *RIN*, *TDR4*, and *MBP7* affected ABA biosynthesis and phenolic acid metabolism. *RIN* knockout increased ABA content, while *TDR4* and *MBP7* knockout increased phenolic acid levels. 4. **TFs Control Steroidal Glycoalkaloid Accumulation**: *RIN*, *TDR4*, and *MBP7* also influenced the accumulation of steroidal glycoalkaloids (SGAs). Mutations in these genes led to increased SGA levels, suggesting their role in SGA biosynthesis. 5. **TFs Differentially Regulate Gene Expression**: The study identified distinct gene expression patterns in mutants compared to WT, highlighting the complex regulatory networks involved in fruit ripening. 6. **TFs Affect Fruit Color and Quality**: Mutations in *TAGL1* and *RIN* resulted in altered fruit colors, providing insights into the genetic basis of color variation in tomatoes. The findings provide a comprehensive understanding of how specific TFs regulate tomato fruit ripening and metabolite accumulation, offering potential targets for breeding tomatoes with desired colors, flavors, and nutritional profiles.This study investigates the regulatory roles of transcription factors (TFs) in tomato fruit ripening and metabolite accumulation. Using CRISPR/Cas9-mediated mutagenesis, the authors generated genetically engineered tomato lines with altered TF expression and analyzed their effects on fruit metabolites and transcript profiles. Key findings include: 1. **TFs Regulate Carotenoid Content**: Mutations in specific TFs, such as *HY5*, *AP2a*, and *TAGL1*, significantly altered carotenoid contents in tomato fruits. For example, *HY5* knockout increased carotenoid content by 30%, while *AP2a* knockout decreased it by 38%. 2. **TFs Influence Sugar Accumulation**: The *HY5* mutant showed a 20% increase in fructose and glucose content compared to wild-type (WT) fruits. Further analysis revealed that *HY5* directly binds to the *SWEET12c* promoter, activating its expression and modulating sugar transport. 3. **TFs Modulate ABA and Phenolic Acid Biosynthesis**: Mutations in *RIN*, *TDR4*, and *MBP7* affected ABA biosynthesis and phenolic acid metabolism. *RIN* knockout increased ABA content, while *TDR4* and *MBP7* knockout increased phenolic acid levels. 4. **TFs Control Steroidal Glycoalkaloid Accumulation**: *RIN*, *TDR4*, and *MBP7* also influenced the accumulation of steroidal glycoalkaloids (SGAs). Mutations in these genes led to increased SGA levels, suggesting their role in SGA biosynthesis. 5. **TFs Differentially Regulate Gene Expression**: The study identified distinct gene expression patterns in mutants compared to WT, highlighting the complex regulatory networks involved in fruit ripening. 6. **TFs Affect Fruit Color and Quality**: Mutations in *TAGL1* and *RIN* resulted in altered fruit colors, providing insights into the genetic basis of color variation in tomatoes. The findings provide a comprehensive understanding of how specific TFs regulate tomato fruit ripening and metabolite accumulation, offering potential targets for breeding tomatoes with desired colors, flavors, and nutritional profiles.