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

Abstract

SUMMARYPolyphenol 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 aestivumL.), 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 thePPO1andPPO2paralogous 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 sevenPPO1andPPO2genes in the spring wheat cultivar ‘Fielder’. Five of the seven edited T1lines exhibited significant reductions in PPO activity, and T2lines had PPO activity up to 86.7% lower than wild-type controls. In the elite winter wheat cultivars ‘Guardian’ and ‘Steamboat’, which have fivePPO1andPPO2genes, PPO activity was reduced by >90% in both T1and T2lines. 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.