Simplex and multiplex CRISPR/Cas9-mediated knockout of grain protease inhibitors in model and commercial barley improves hydrolysis of barley and soy storage proteins

Anti-nutritional factors in plant seeds diminish the utilization of nutrients in feed and food. Among these, protease inhibitors inhibit protein degradation by exogenous proteases during digestion. Through conventional and selection-gene-free genome editing using ovules as explants, we used simplex and multiplex CRISPR/Cas9 for studying the impact of chymotrypsin inhibitor CI-1A, CI-1B and CI-2, Bowman-Birk trypsin inhibitor, Serpin-Z4, and barley ɑ-amylase/subtilisin inhibitor on barley and soybean storage protein degradation. Mutants were generated in the commercial cultivar Stairway, having a high level of protease inhibition, and the barley model cultivar Golden Promise, having a lower inhibition level. In Golden Promise, all individual knockouts decreased the inhibition of the three proteases α-chymotrypsin, trypsin and the commercial feed protease Ronozyme ProAct significantly. The triple knockout of all chymotrypsin inhibitors further decreased the inhibition of α-chymotrypsin and Ronozyme ProAct proteases. Degradations of recombinant barley storage proteins B- and C-hordeins were significantly improved following mutagenesis. In Stairway, a single knockout of CI-1A almost compares to the effect on the proteases achieved for the triple knockout in Golden promise, uncovering CI-1A as the major protease inhibitor in that cultivar. The Stairway mutant demonstrated significantly improved degradation of recombinant barley hordeins and in the soybean storage proteins glycinin and β-conglycinin. The results of this study provide insights into cereal protease inhibitor genes and their negative effects on the degradation of barley storage protein and the most important plant protein from soybeans. The study suggests a future focus on plant protease inhibitors as a major target for improving feed and food protein digestibility.