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

Abstract

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.

CRISPR-Cas: A robust technology for enhancing consumer-preferred commercial traits in crops

“…Heritable genomic alterations and transgene-free plants were produced as a consequence of the targeted plant genome editing made possible by CRISPR-Cas9 ( Hu and Gao, 2023 ). Utilizing the CRISPR-Cas system several crops have shown both qualitative and quantitative gains ( Zhang et al., 2020 ; Carrijo et al., 2021 ; Confalonieri et al., 2021 ; Eid et al., 2021 ; Oz et al., 2021 ; Zhang et al., 2021 ; Biswas et al., 2022 ; Lu and Tian, 2022 ). An overview of CRISPR- Cas technological advancements are shown in Figure 2 .…”

Engineering the plant metabolic system by exploiting metabolic regulation

“…Also, in other crops like soybean, the simultaneous CRISPR‐based editing of GmF3H1 , GmF3H2 , and GmFNSII‐1 was carried out to increase its isoflavone content and thus improve its resistance to the soybean mosaic virus (Zhang et al, 2020). In Medicago truncatula , rerouting of the saponin pathway was achieved by knocking out the MtCYP93E2 and MtCYP72A61 genes, resulting in the accumulation of hemolytic sapogenins and avoiding the accumulation of sapogenols (Confalonieri et al., 2021).…”

Gene-Editing Technologies and Applications in Legumes: Progress, Evolution, and Future Prospects

“… Rodas et al (2021) edited the M. truncatula SUPERMAN ( MtSUP ) gene with CRISPR/Cas 9 and determined the impairment of MtSUP function with observing defects in floral development and inflorescence architecture in mtsup mutant allel carrying plants. Confalonieri et al (2021) also used the CRISPR/Cas9 gene-editing tool to knock out the two cytochrome P 450 genes ( CYP93E2 and CYP72A61 ) that are responsible for soyasapogenol B production in Medicago spp. Their results showed that 51 putative CYP93E2 mutant plant lines with an 84% editing efficiency did not produce soyasapogenols in the leaves, stems, and roots with diverting the metabolic pathway toward the production of valuable hemolytic sapogenins.…”