Data_Sheet_2_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）是一类兼具多种生物活性与药用价值的活性化合物，已得到广泛研究。本研究采用基于CRISPR/Cas9的技术体系，设计两条单一位点向导RNA（single-site guide RNAs），在蒺藜苜蓿（Medicago truncatula，又称桶状苜蓿barrel medic）中对负责大豆皂苷元B（soyasapogenol B）生物合成的CYP93E2与CYP72A61基因进行敲除；其中大豆皂苷元B是苜蓿属植物中含量最丰富的大豆皂苷元（soyasapogenol）。实验未获得靶标CYP72A61基因发生突变的转基因植株，而通过根癌农杆菌（Agrobacterium tumefaciens）介导的遗传转化，共获得52株推定的CYP93E2突变株系。其中对51株株系进行测序，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类型。研究进一步选取4株T0代突变株系，在温室条件下检测其皂苷元含量与植株生长表现。结果表明，所有被测CYP93E2敲除突变体的叶片、茎秆与根系均无法合成大豆皂苷元，而是将代谢流转向生成高价值的溶血皂苷元（hemolytic sapogenins）。在温室栽培条件下，CYP93E2突变体的植株形态未受到不良影响。此外，与对照植株相比，CYP93E2突变体中皂苷生物合成通路基因的表达存在显著差异。本研究结果为利用CRISPR/Cas9技术对植物源高价值化合物进行代谢工程改造提供了全新的重要见解，同时也为探究皂苷在植物体内（in planta）的生理功能提供了可靠的研究基础。