Sci Rep. 2017 Mar 3;7:43320. doi: 10.1038/srep43320.
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.
Figure 2: Targeted genome modification using the CRISPR/Cas9 system.
(A) The Cas9 expression cassette is driven by the 2X35S promoter. The gRNA is driven by the A. thaliana U6-26 promoter. The hygromycin resistance gene is driven by CaMV 35S. (B) Upper: Gene structure of SmCPS1 and the location of three gRNAs. Numbers 1–13 indicate exons. The location of sgRNA1, sgRNA2, and sgRNA3 are indicated by red lines and by black arrows in exons 1, 4, and 11. “CGG”, “TGG”, and “GGG” sequences are, respectively, the PAMs of sgRNA1, sgRNA2, and sgRNA3. Lower: Sequences amplified from genomic DNA isolated from primary transgenic hairy root lines of sgRNA3 derivatives. The WT sequence appears at the top, with the PAM sequence shown in blue and the target sequence highlighted in yellow. DNA insertions, point mutations are shown as red letters. Deletions are shown as dashes: + : insertion, −: deletion. The indel size shows the loss/gain in amplicon length in target loci. The WT sequence is not listed for each line. (C) Denaturing PAGE separation. A silver-stained denaturing PAGE separation showing the DNA fragments generated from WT (wild type) and different mutant root lines. M: 100 bp DNA marker. (D) The appearance of wild type and homozygous mutants (No. 1, No. 9, No. 32) roots. The WT produces a red periderm while No. 1, No. 9, and No. 32 are white. Bar = 1 cm.