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http://dx.doi.org/10.1016/j.jgr.2021.06.004

Modification of ginsenoside saponin composition via the CRISPR/Cas9-mediated knockout of protopanaxadiol 6-hydroxylase gene in Panax ginseng  

Choi, Han Suk (Division of Forest Sciences, College of Forest and Environmental Sciences, Kangwon National University)
Koo, Hyo Bin (Division of Forest Sciences, College of Forest and Environmental Sciences, Kangwon National University)
Jeon, Sung Won (Division of Forest Sciences, College of Forest and Environmental Sciences, Kangwon National University)
Han, Jung Yeon (Division of Forest Sciences, College of Forest and Environmental Sciences, Kangwon National University)
Kim, Joung Sug (Department of Biosciences and Bioinformatics, Myongji University)
Jun, Kyong Mi (Plant Molecular Genetics Institute, GreenGene Biotech Inc.)
Choi, Yong Eui (Division of Forest Sciences, College of Forest and Environmental Sciences, Kangwon National University)
Publication Information
Journal of Ginseng Research / v.46, no.4, 2022 , pp. 505-514 More about this Journal
Abstract
Background: The roots of Panax ginseng contain two types of tetracyclic triterpenoid saponins, namely, protopanaxadiol (PPD)-type saponins and protopanaxatiol (PPT)-type saponins. In P. ginseng, the protopanaxadiol 6-hydroxylase (PPT synthase) enzyme catalyses protopanaxatriol (PPT) production from protopanaxadiol (PPD). In this study, we constructed homozygous mutant lines of ginseng by CRISPR/Cas9-mediated mutagenesis of the PPT synthase gene and obtained the mutant ginseng root lines having complete depletion of the PPT-type ginsenosides. Methods: Two sgRNAs (single guide RNAs) were designed for target mutations in the exon sequences of the two PPT synthase genes (both PPTa and PPTg sequences) with the CRISPR/Cas9 system. Transgenic ginseng roots were generated through Agrobacterium-mediated transformation. The mutant lines were screened by ginsenoside analysis and DNA sequencing. Result: Ginsenoside analysis revealed the complete depletion of PPT-type ginsenosides in three putative mutant lines (Cr4, Cr7, and Cr14). The reduction of PPT-type ginsenosides in mutant lines led to increased accumulation of PPD-type ginsenosides. The gene editing in the selected mutant lines was confirmed by targeted deep sequencing. Conclusion: We have established the genome editing protocol by CRISPR/Cas9 system in P. ginseng and demonstrated the mutated roots producing only PPD-type ginsenosides by depleting PPT-type ginsenosides. Because the pharmacological activity of PPD-group ginsenosides is significantly different from that of PPT-group ginsenosides, the new type of ginseng mutant producing only PPD-group ginsenosides may have new pharmacological characteristics compared to wild-type ginseng. This is the first report to generate target-induced mutations for the modification of saponin biosynthesis in Panax species using CRISPR-Cas9 system.
Keywords
CRISPR/Cas9 system; construction of mutant; genetic transformation; ginsenoside; protopanaxadiol 6-hydroxylase; saponins; sgRNA; triterpene;
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