title: 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.
authors.affiliation: Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.|Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China.
authors.forename: Bin
authors.initials: B
authors.lastname: Li
authors.affiliation: National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, 100700, China.
authors.forename: Guanghong
authors.initials: G
authors.lastname: Cui
authors.affiliation: Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China.
authors.forename: Guoan
authors.initials: G
authors.lastname: Shen
authors.affiliation: National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, 100700, China.
authors.forename: Zhilai
authors.initials: Z
authors.lastname: Zhan
authors.affiliation: National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, 100700, China.
authors.forename: Luqi
authors.initials: L
authors.lastname: Huang
authors.affiliation: Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
authors.forename: Jiachun
authors.initials: J
authors.lastname: Chen
authors.affiliation: Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China.
authors.forename: Xiaoquan
authors.initials: X
authors.lastname: Qi
mesh_terms: D045784:Abietanes; D060054:Agrobacterium / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D064113:CRISPR-Cas Systems; D004224:Diterpenes / Q000378:metabolism; D000072669:Gene Editing / Q000379:methods; D018506:Gene Expression Regulation, Plant; D055786:Gene Knockout Techniques; D018745:Genome, Plant; D062385:Hydroxybenzoates / Q000378:metabolism; D008025:Ligases / Q000172:deficiency / Q000235:genetics*; D016296:Mutagenesis; D010616:Phenanthrenes; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D018517:Plant Roots / Q000235:genetics* / Q000378:metabolism; D010946:Plants, Medicinal; D027561:Salvia miltiorrhiza / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D045784:Abietanes; D004224:Diterpenes; D062385:Hydroxybenzoates; D010616:Phenanthrenes; D010940:Plant Proteins; C021751:tanshinone; C037886:cryptotanshinone; D008025:Ligases; C017616:phenolic acid