Tanshinone accumulation serves as a critical determinant of medicinal value in Salvia miltiorrhiza cultivars. Precise fine-tuning of tanshinone biosynthesis while preserving elite agronomic traits remains a pivotal challenge in molecular breeding. Here, we report, for the first time, the successful application of CRISPR/Cas9-mediated upstream open reading frame (uORF) editing in medicinal plants to enhance the production of specialized metabolites. Five evolutionarily conserved uORFs identified in the 5′ leader sequence of the key diterpene synthase gene SmCPS1 were strategically edited to modulate post-transcriptional regulation. Homozygous mutants engineered through precision gene editing exhibited 1.19- to 1.81-fold enhanced tanshinone accumulation compared to the controls, correlating with coordinated transcriptional activation of core biosynthetic genes (SmHMGR1, SmKSL1, SmCYP76AH1, SmCYP76AH3). Integrative molecular analyses demonstrated unchanged SmCPS1 transcript levels and enhanced protein accumulation, mechanistically confirming uORF-mediated translational potentiation of the cognate main ORF. This study establishes uORF engineering as a robust platform for predictable metabolic engineering in S. miltiorrhiza plants. Future applications could expand this strategy to uORFs of rate-limiting enzymes or transcriptional regulators, enabling multidimensional optimization of high-value metabolites in medicinal species.

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