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

Han Suk Choi; Hyo Bin Koo; Sung Won Jeon; Jung Yeon Han; Joung Sug Kim; Kyong Mi Jun; Yong Eui Choi

doi: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

J Ginseng Res. 2022 Jul;46(4):505-514. doi: 10.1016/j.jgr.2021.06.004. Epub 2021 Jun 18.

Authors

Han Suk Choi^{1

2}, Hyo Bin Koo^{1

2}, Sung Won Jeon^{1

2}, Jung Yeon Han¹, Joung Sug Kim³, Kyong Mi Jun⁴, Yong Eui Choi^{1

2}

Affiliations

¹ Division of Forest Sciences, College of Forest and Environmental Sciences, Kangwon National University, Chuncheon, Republic of Korea.
² Department of Bio-Health Convergence, Graduate School, Kangwon National University, Chuncheon-si, Republic of Korea.
³ Department of Biosciences and Bioinformatics, Myongji University, Yongin-si, Republic of Korea.
⁴ Plant Molecular Genetics Institute, GreenGene Biotech Inc., Yongin-si, Republic of Korea.

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; CRISPR/Cas9, clustered regularly interspaced short palindromic repeats associated with nuclease Cas9; CYP, cytochrome P450; RNAi, RNA interference; RT-PCR, reverse transcription-PCR; construction of mutant; genetic transformation; ginsenoside; protopanaxadiol 6-hydroxylase; qPCR, quantitative real-time RT-PCR; saponins; sgRNA; sgRNA, single guide RNA; triterpene.