Editing of an Alpha-Kafirin Gene Family Increases, Digestibility and Protein Quality in Sorghum

Abstract

Kafirins are the major storage proteins in sorghum (<i>Sorghum bicolor</i>) grains and form protein bodies with poor digestibility. Since kafirins are devoid of the essential amino acid lysine, they also impart poor protein quality to the kernel. The α-kafirins, which make up most of the total kafirins, are largely encoded by the <i>k1C</i> family of highly similar genes. We used a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing approach to target the <i>k1C</i> genes to create variants with reduced kafirin levels and improved protein quality and digestibility. A single guide RNA was designed to introduce mutations in a conserved region encoding the endoplasmic reticulum signal peptide of α-kafirins. Sequencing of kafirin PCR products revealed extensive edits in 25 of 26 events in one or multiple <i>k1C</i> family members. T1 and T2 seeds showed reduced α-kafirin levels, and selected T2 events showed significantly increased grain protein digestibility and lysine content. Thus, a single consensus single guide RNA carrying target sequence mismatches is sufficient for extensive editing of all <i>k1C</i> genes. The resulting quality improvements can be deployed rapidly for breeding and the generation of transgene-free, improved cultivars of sorghum, a major crop worldwide.