Data_Sheet_7_CRISPR/Cas9-Mediated Targeted Mutagenesis of CYP93E2 Modulates the Triterpene Saponin Biosynthesis in Medicago truncatula.PDF

In the Medicago genus, triterpene saponins are a group of bioactive compounds extensively studied for their different biological and pharmaceutical properties. In this work, the CRISPR/Cas9-based approach with two single-site guide RNAs was used in Medicago truncatula (barrel medic) to knock-out the CYP93E2 and CYP72A61 genes, which are responsible for the biosynthesis of soyasapogenol B, the most abundant soyasapogenol in Medicago spp. No transgenic plants carrying mutations in the target CYP72A61 gene were recovered while fifty-two putative CYP93E2 mutant plant lines were obtained following Agrobacterium tumefaciens-mediated transformation. Among these, the fifty-one sequenced plant lines give an editing efficiency of 84%. Sequencing revealed that these lines had various mutation patterns at the target sites. Four T0 mutant plant lines were further selected and examined for their sapogenin content and plant growth performance under greenhouse conditions. The results showed that all tested CYP93E2 knock-out mutants did not produce soyasapogenols in the leaves, stems and roots, and diverted the metabolic flux toward the production of valuable hemolytic sapogenins. No adverse influence was observed on the plant morphological features of CYP93E2 mutants under greenhouse conditions. In addition, differential expression of saponin pathway genes was observed in CYP93E2 mutants in comparison to the control. Our results provide new and interesting insights into the application of CRISPR/Cas9 for metabolic engineering of high-value compounds of plant origin and will be useful to investigate the physiological functions of saponins in planta.

在苜蓿属（Medicago）植物中，三萜皂苷（triterpene saponins）是一类被广泛研究的生物活性化合物，其具备多样的生物学与药学特性。本研究采用基于CRISPR/Cas9、搭载两条单一位点向导RNA的基因编辑技术，对蒺藜苜蓿（Medicago truncatula，俗称barrel medic）中负责大豆皂醇B生物合成的CYP93E2与CYP72A61基因进行敲除——大豆皂醇B是苜蓿属（Medicago spp.）植物中含量最丰富的大豆皂醇。
研究未获得携带CYP72A61靶基因突变的转基因植株，但经农杆菌（Agrobacterium tumefaciens）介导转化后，共获得52个推定的CYP93E2突变株系。其中，对51个株系进行测序，编辑效率达84%。测序结果显示，这些株系的靶位点存在多种突变模式。
选取4个T0代突变株系，在温室条件下检测其皂醇含量与植株生长表现。结果表明，所有受试的CYP93E2敲除突变体的叶片、茎秆与根系中均未合成大豆皂醇，而是将代谢流转向生成高价值的溶血皂醇。温室条件下，CYP93E2突变体的植株形态未受到负面影响。此外，与对照组相比，CYP93E2突变体的皂苷生物合成通路基因存在差异表达。
本研究结果为利用CRISPR/Cas9技术开展植物来源高价值化合物的代谢工程研究提供了全新的重要见解，同时可为探究皂苷在植株体内的生理功能提供重要参考。