CRISPR/Cas9-mediated targeted mutagenesis of GmMYB100 to increase isoflavone content in soybean seeds.

UNLABELLED: CRISPR/Cas9-mediated targeted mutagenesis of GmMYB100 was used to generate genome-edited soybean (Glycine max (L.) Merrill cv. JS335) lines with enhanced isoflavone accumulation. A single guide RNA targeting the first exon of GmMYB100 was cloned into the pHSE401 vector and introduced into soybean via Agrobacterium tumefaciens strain EHA105. Molecular characterization of stable T0 plants confirmed the integration of the Cas9 and hptII genes, while sequence analysis identified insertion/deletion (indel) mutations at the target site, confirming successful genome editing. The edited lines (T0) showed significantly increased isoflavone accumulation, with daidzein and genistein contents increasing by 2.85-fold and 1.23-fold, respectively, compared with non-transformed plants. Quantitative RT-PCR analysis revealed significant upregulation of major isoflavone biosynthetic genes in immature cotyledons, mature cotyledons, and seeds, consistent with the enhanced metabolite accumulation. These results demonstrate that disruption of GmMYB100 relieves negative regulation of the isoflavonoid biosynthetic pathway, leading to increased isoflavone production in soybean seeds. The study establishes GmMYB100 as an effective target for CRISPR/Cas9-mediated metabolic engineering and provides a practical strategy for improving the nutritional quality of soybean through precise genome editing. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at https://doi.org/10.1007/s13205-026-05033-1.