A barley starch synthase IIa (ssIIa) null mutant has been commercialized as a healthy grain of its benefits for human health; however, it has a shrunken grain and reduced yield. Here, we used a multiplex genome-editing system to produce single, double, and triple null mutations of five barley starch synthesis enzyme genes for either increasing the content of beneficial grain ingredients or increasing the grain yield compared to ssIIa mutant.

The grain ingredients of these mutants were analyzed, and the ssIIa null mutant exhibited the highest content of β-glucan. The sbeIIasbeIIb and ssIIasbeIIasbeIIb mutants produced amylose and resistant starch over 86% and 12%, respectively, which were extremely higher than that in the ssIIa mutant. Furthermore, these mutants have greater 1000-grain weight, and less fructan and dietary fiber contents than those of the ssIIa mutant.

The ssIIassIVa mutant produced similar resistant starch content and 1000-grain weight, higher levels of fructan compared to the ssIIa mutant. The ssIIassIIIassIVa mutant had a similar 1000-grain weight, and less resistant starch but more fructan and dietary fiber than the ssIIa mutant. The ssIIassIIIa mutant had a greater 1000-grain weight, similar resistant starch content, and lower levels of fructan, and dietary fiber compared to the ssIIa mutant.

Three mutants, ssIIassIVa, sbeIIasbeIIb and ssIIasbeIIasbeIIb, exhibited improved levels of amylose and dietary fibre and/or a higher grain weight compared with the single ssIIa mutant. These were determined to be the best choices among the five polygenic mutants for future applications in the production of healthy food products.

Our study demonstrates that Cas9-mediated multiplex gene editing is feasible for modifying starch to generate grain with higher RS contents than targeted editing of single genes and provides a theoretical basis and genetic resource for breeding barley with improved health benefits.