Multiplex genome editing targeting soybean with ultra-low anti-nutritive oligosaccharides

Soybean is the primary source of plant protein for humans. Owing to the indigestibility of the raffinose family of oligosaccharides (RFO), raffinose and stachyose are considered anti-nutritive factors in soybean seeds. Low-RFO soybean cultivars are generated by mutagenesis of RFO biosynthesis genes, but the carbohydrate profiles invite further modification to lower RFOs. This study employed a pooled multiplex genome editing approach to target four seed-specifically expressed genes mediating RFO biosynthesis, encoding three raffinose synthases (RS2, RS3, and RS4) and one stachyose synthase. In T₁ progeny, rs2/rs3 and rs4/sts homozygous double mutants and a rs2/rs3/rs4/sts quadruple mutant (rfo-4m) were characterized. The rs2/rs3 mutant showed reduced raffinose and stachyose contents, but the rs4/sts mutant showed only reduced stachyose in seeds. The RFO contents in the rfo-4m mutant were almost eliminated. Metabolomic analysis showed that the mutation of four RFO biosynthesis genes led to a shift of metabolic profile in the seeds, including the accumulation of several oligosaccharides-related metabolites. These mutants could contribute to precision breeding of soybean cultivars for soy food production.