Improving digestibility of sorghum proteins by CRISPR/Cas9-based genome editing

Kafirins are the primary storage proteins in sorghum kernels that provide amino acids for seed germination. The highly proteolytic resistant gamma- and beta-kafirins form the cross-linked outer layers that encapsulate alpha-kafirins to generate protein bodies, resulting in poor digestibility of sorghum grains. The sorghum kafirins thus contribute to the poor quality of the kernels. The nutritional quality and digestibility of sorghum grains can be improved by reducing the contents of kafirin. gamma-Kafirin is encoded by the K2G gene, which is located on sorghum chromosome 2. In this study, we used CRISPR/Cas9 gene editing to target the K2G gene to create new sorghum lines with reduced levels of kafirin. A guide RNA (sgRNA) was designed to introduce mutations in the CDS region that encodes the endoplasmic reticulum signal peptide of gamma-kafirin. The pK2GsgRNA/Cas9 vector was transformed into sorghum using the pollen-mediated transformation method. Sequencing of the transformants showed that three out of 24 transgenic plants contain genetic mutations in the targeted region. Compared with the wildtype, the gamma-kafirin contents of the mutant plants decreased by 12.75%-19.22%, and the protein digestibility of the mutant kernels increased by 26.91%-74.31% in raw flour. Although the grain weights remained comparable to those of the wildtype, the growth of the mutant plants was more vigorous as the mutant shoots grew taller and thicker compared with those of the wildtype. Our work advances the ability to improve the digestibility of an important crop. The resulting quality improvements can also be rapidly deployed for breeding and generation of transgene-free, improved cultivars of sorghum, a major crop worldwide.