Development of sorghum mutants with improved in vitro protein digestibility by CRISPR/Cas9 editing of kafirin genes

Sorghum (Sorghum bicolor (L.) Moench) is a major world crop that is a reliable source of fodder and food grain in arid regions. However, unlike other cereals, sorghum grain has low nutritional value, owing mainly to the resistance of its storage proteins (kafirins) to protease digestion. Changing the composition of kafirins or their primary structure may address this problem. To induce mutations in kafirin-encoding genes that were expected to disturb their accumulation in endosperm cells, we used a genome-editing approach. By Agrobacterium-mediated genetic transformation of immature embryos of cv. Avans, we obtained 14 transgenic plants with genetic constructs for site-directed mutagenesis of the k1C5 and gKAF1 genes encoding 22 kDa α- and 28 kDa γ-kafirins, respectively. Sequencing of 5 regenerants obtained by using k1C5-addressing vector revealed two plants with mutations. T₁ progeny of these mutants had higher in vitro digestibility of endosperm proteins (86%–92%), in comparison with the donor Avans (63%–67%). The kernels of these plants had a thick vitreous endosperm. A mutant with increased in vitro protein digestibility and vitreous endosperm, carrying a mutation in the target sequence, was also obtained by use of the gKAF1-addressing vector. Thus, using genome editing technology, we have obtained mutants with improved kafirin digestibility that can be used in sorghum breeding.