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

• DOI: 10.3389/fpls.2021.690231
• Publication Date: 2021-07-26T11:46:00Z
• Authors: Massimo Confalonieri; Maria Carelli; Silvia Gianoglio; Andrea Moglia; Elisa Biazzi; Aldo Tava
• Keywords: Medicago truncatula; Sapogenin; Medicago; Lotus japonicus; Medicago sativa

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