Multi-target genome editing reduces polyphenol oxidase activity in wheat (Triticum aestivum L.) grains

Polyphenol oxidases (PPO) are dual activity metalloenzymes that catalyse the production of quinones. In plants, PPO activity may contribute to biotic stress resistance and secondary metabolism but is undesirable for food producers because it causes the discolouration and changes in flavour profiles of products during post-harvest processing. In wheat (Triticum aestivum L.), PPO released from the aleurone layer of the grain during milling results in the discolouration of flour, dough, and end-use products, reducing their value. Loss-of-function mutations in the PPO1 and PPO2 paralogous genes on homoeologous group 2 chromosomes confer reduced PPO activity in the wheat grain but limited natural variation and small intergenic distances between these genes complicates the selection of extremely low-PPO wheat varieties by recombination. In the current study, a CRISPR/Cas9 construct with one single guide RNA (sgRNA) targeting a conserved copper binding domain was used to edit all seven PPO1 and PPO2 genes in the spring wheat cultivar Fielder. Five of the seven edited T1 lines exhibited significant reductions in PPO activity, and T2 lines had PPO activity up to 86.7% lower than wild-type controls. In the elite winter wheat cultivars Guardian and Steamboat, which have five PPO1 and PPO2 genes, PPO activity was reduced by >90% in both T1 and T2 lines. This study demonstrates that multi-target editing at late stages of variety development could complement selection for beneficial alleles in crop breeding programmes by inducing novel variation in loci inaccessible to recombination.