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Polyphenol oxidases (PPOs) catalyze the oxidization of polyphenols, which in turn causes the browning of the eggplant berry flesh after cutting. This has a negative impact on fruit quality for both industrial transformation and fresh consumption. Ten  PPO  genes (named  SmelPPO1 - 10 ) were identified in eggplant thanks to the recent availability of a high-quality genome sequence. A CRISPR/Cas9-based mutagenesis approach was applied to knock-out three target  PPO  genes ( SmelPPO4, SmelPPO5 , and  SmelPPO6) , which showed high transcript levels in the fruit after cutting. An optimized transformation protocol for eggplant cotyledons was used to obtain plants in which Cas9 is directed to a conserved region shared by the three  PPO  genes. The successful editing of the  SmelPPO4, SmelPPO5 , and  SmelPPO6  loci of  in vitro  regenerated plantlets was confirmed by Illumina deep sequencing of amplicons of the target sites. Besides, deep sequencing of amplicons of the potential off-target loci identified  in silico  proved the absence of detectable non-specific mutations. The induced mutations were stably inherited in the T 1  and T 2  progeny and were associated with a reduced PPO activity and browning of the berry flesh after cutting. Our results provide the first example of the use of the CRISPR/Cas9 system in eggplant for biotechnological applications and open the way to the development of eggplant genotypes with low flesh browning which maintain a high polyphenol content in the berries.

Simultaneous CRISPR/Cas9 Editing of Three PPO Genes Reduces Fruit Flesh Browning in Solanum melongena L.