Rapid and Efficient Creation of Sweet–Waxy Maize Germplasm via CRISPR/Cas9-Mediated Gene Editing of Sh2 and Wx

Sweet–waxy maize is a highly valuable specialty maize type with an increasing market demand, but conventional breeding methods for producing sweet–waxy maize are restricted by severe bottlenecks, such as long breeding cycles and linkage drag. This study was conducted to rapidly create sweet–waxy maize germplasm using CRISPR/Cas9 genome-editing technology. We used a CRISPR/Cas9 system to target maize Sh2 (regulating the super-sweet kernel trait) and Wx (controlling the waxy kernel trait), which are two key genes in the starch biosynthesis pathway. Two small-guide RNAs (sgRNAs) designed for each gene were incorporated into CRISPR/Cas9 vectors, which were then introduced into maize via Agrobacterium-mediated transformation. We obtained Cas9-free T3 homozygous sh2 and wx mutant lines with significant increases in kernel soluble sugar and amylopectin contents, respectively, but no adverse changes to major agronomic traits. Using these Cas9-free lines, we developed a new type of sweet–waxy maize germplasm, in which waxy and sweet kernels on the same ear segregated at a 3:1 ratio. Our results indicate that CRISPR/Cas9-mediated editing of Sh2 and Wx can efficiently generate sweet–waxy maize germplasm with no detectable linkage drag. The study methods would be useful for optimizing the molecular breeding of novel and innovative maize germplasm.