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

Abstract

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 <i>PPO</i> genes (named <i>SmelPPO1</i>-<i>10</i>) 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 <i>PPO</i> genes (<i>SmelPPO4, SmelPPO5</i>, and <i>SmelPPO6)</i>, 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 <i>PPO</i> genes. The successful editing of the <i>SmelPPO4, SmelPPO5</i>, and <i>SmelPPO6</i> loci of <i>in vitro</i> 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 <i>in silico</i> proved the absence of detectable non-specific mutations. The induced mutations were stably inherited in the T₁ and T₂ 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.