CRISPR/Cas9-mediated uORF engineering enhances tanshinone biosynthesis in Salvia miltiorrhiza

Abstract

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.

Acknowledgements

This work was supported by the Scientific and Technological Project of China Academy of Chinese Medical Sciences (CI2024C005YN, CI2023E002), the Sustainable Utilization Capacity Building Project for Valuable Chinese Medicinal Resources (2060302), the National Key R&D Program of China (2018YFA0900600), and grants from the Shanghai Jiao Tong University Transmed Awards Program (20190104).

Authors contributions

J.S., L.H., and K.T. conceived and designed the study. J.S., B.P., H.Z., and L.L. conducted most of the experimental work, with B.P., D.L., X.H., and L.L. performing data analysis. The manuscript was drafted by J.S. and K.T., and subsequently reviewed and approved by all authors.

Data availability

The data that support the findings of this study are available in the supplementary material of the article.

Conflicts of interest statement

The authors declare no competing interests.

Supplementary material

Supplementary material is available at Horticulture Research online.

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