CRISPR-knockout of β-kafirin in sorghum does not recapitulate the grain quality of natural mutants

Main conclusionWhen gene editing was applied to knockout beta-kafirin, there was a compensatory increase of gamma-kafirin which does not occur in domesticated null varieties, so enhanced grain quality was not achieved. Sorghum bicolor is an important animal feedstock cereal crop throughout Australia and the southern United States, where its use as a food product is limited by issues with low calorific and nutritive value. Qualities such as reduced digestibility and low essential amino acid content are directly attributed to the kafirin grain storage proteins, the major components of protein bodies within the endosperm. Specifically, the beta- and gamma-kafirins have few protease cleavage sites and high levels of cysteine residues which lead to a highly cross-linked shell of intra- and inter-molecular disulphide linkages that encapsulate the more digestible alpha- and delta-kafirins in the core of the protein bodies. Naturally occurring beta-kafirin mutants exist and are known to have improved grain quality, with enhanced protein contents and digestibility, traits which are often attributed to the lack of this cysteine-rich kafirin in the mature grain. However, when CRISPR/Cas9 editing was used to create beta-kafirin knockout lines, there was no improvement to grain quality in the Tx430 background, although they did have unique protein composition and changes to protein body morphology in the vitreous endosperm. One explanation of the divergence in quality traits found the lines lacking beta-kafirin are due to a drastic increase of gamma-kafirin which was only found in the gene edited lines. This study highlights that in some germplasm, there is a level of redundancy between the peripheral kafirins, and that improvement of grain protein digestibility cannot be achieved by simply removing the beta-kafirin protein in all genetic backgrounds.

Automated summary

This study shows that knocking out beta-kafirin gene does not improve grain quality in sorghum. Instead, there is a compensatory increase in gamma-kafirin gene, which leads to no enhancement in grain quality.