Targeted mutagenesis in the medicinal plant Salvia miltiorrhiza

Abstract

CRISPR/Cas9 is a powerful genome editing tool that has been extensively used in model plants and crops, such as Arabidopsis thaliana, rice, wheat, and soybean. Here, we report the use of CRISPR/Cas9 to precisely knock out the committed diterpene synthase gene (SmCPS1) involved in tanshinone biosynthesis in Salvia miltiorrhiza, a traditional Chinese medicinal herb with significant pharmacological activities, such as vasorelaxation, protection against ischemia-reperfusion injury, and antiarrhythmic effects. Three homozygous and eight chimeric mutants were obtained from 26 independent transgenic hairy root lines by Agrobacterium rhizogenes-mediated transformation. The metabolomic analysis based on LC-qTOF-MS and Q-TRAP-LC-MS/MS revealed that tanshinones, especially cryptotanshinone, tanshinone IIA and tanshinone I, are completely missing in homozygous mutants, without influencing other phenolic acid metabolites. By contrast, tanshinones are decreased but still detectable in chimeric mutants, which is similar to a previously-reported an RNAi study of SmCPS1. These results demonstrate that Agrobacterium rhizogenes- mediated transformation using CRISPR/Cas9 is a simple and efficient genome editing tool in S. miltiorrhiza, thus paving the way for large-scale genome editing in S. miltiorrhiza, which is important for pathway elucidation of secondary metabolites, quality improvement, and yield increases for this valuable traditional Chinese medicinal herb.

Multiplexed CRISPR/Cas9‐mediated metabolic engineering of γ‐aminobutyric acid levels in Solanum lycopersicum

“…Li et al . () used CRISPR/Cas9 techniques that targeted the committed diterpene synthase gene ( SmCPS1 ) involved in tanshinone biosynthesis in Salvia miltiorrhiza , and different tanshinone accumulation levels were detected in chimeric and homozygous mutants. Therefore, these results suggested that SmCPS1 in chimeric mutants was knocked down instead of being knocked‐out.…”

“…Both GABA-2 (CR-GABA-20) and GABA-3 (CR-GABA-56) mutants were chimeric ( Figure S3), and chimeric mutations might not result in the complete loss of gene function, and the GABA-TP enzyme activity in the fruits of these two GABA mutants remained although the level was significantly lower than that of WT plants (Figure 8). Li et al (2017) used CRISPR/Cas9 techniques that targeted the committed diterpene synthase gene (SmCPS1) involved in tanshinone biosynthesis in Salvia miltiorrhiza, and different tanshinone accumulation levels were detected in chimeric and homozygous mutants. Therefore, these results suggested that SmCPS1 in chimeric mutants was knocked down instead of being knocked-out.…”

Ascertaining the Paradigm of Secondary Metabolism Enhancement through Gene Level Modification in Therapeutic Plants

“…Li et al 2017 [65] Salvia miltiorrhiza phenolic acid biosynthetic pathway rosmarinic acid synthase gene (SmRAS) CRISPR/Cas9 Zhou et al 2018 [66] Solanum lycopersicum…”

Biosynthesis of bioactive ingredients of Salvia miltiorrhiza and advanced biotechnologies for their production

“…CRISPR/Cas9 is a gene-editing tool gaining popularity in recent years. Li et al [72] knocked out the committed diterpene synthase gene (SmCPS1) involved in tanshinone biosynthesis in S. miltiorrhiza by CRISPR/ Cas9. The metabolomic analysis revealed that tanshinones, especially cryptotanshinone, TA-IIA and tanshinone I, are completely missing in homozygous mutants, without influencing other phenolic acid metabolites.…”