CRISPR/Cas9 Knock-out of Polyphenol Oxidase Genes for Improving the Quality of Eggplant Fruits

Eggplant (Solanum melongena) fruits are rich in antioxidant phenolic compounds which, after cutting, become available to polyphenol oxidase enzymes (PPOs). The latter catalyze the oxidation of phenolic compounds and cause browning of the fruit flesh to the detriment of berry quality for both fresh consumption and industrial transformation. Commercial varieties characterized by low flesh browning have been the result of indirect selection for low content in phenolics, and thus reduced nutraceutical properties. Ten PPO genes (named PPO1-10) were isolated and characterized, also thanks to the recent availability of a high quality and annotated eggplant genome sequence. Their qPCR expression profiles were assessed in the fruit flesh and peel of three eggplant varieties, immediately and 30 min after cutting. Increases of transcript levels after cutting were highlighted in PPO1, PPO3, PPO4 and PPO5 genes. A CRISPR/Cas9 system, integrated into the GoldenBraid (GB) cloning standard tool, was applied to knock-out the four PPO genes. The GB CRISPR Cas9 vectors carried 2 guide RNAs: one targeting PPO1 and 3, the other PPO4 and 5 as well as PPO6 (due to the high homology between these gene family members). On the basis of both polyphenol content and in vitro regeneration potential, the three eggplant varieties were selected for Agrobacterium-mediated transformation. Mutations at the target sites as well as off-targets effects were assessed by Next-Generation Sequencing in genomic DNA extracted from in vitro regenerated shoots.