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

• Published in: Frontiers in plant science 2020-12, Vol.11, p.607161
• Main Authors: Maioli, Alex, Gianoglio, Silvia, Moglia, Andrea, Acquadro, Alberto, Valentino, Danila, Milani, Anna Maria, Prohens, Jaime, Orzaez, Diego, Granell, Antonio, Lanteri, Sergio, Comino, Cinzia
• Format: Article
• Language: English
• Subjects: CRISPR/Cas 9; eggplant; gene editing; knock-out; Plant Science; polyphenol oxydase
• ISSN: 1664-462X; 1664-462X
• DOI: 10.3389/fpls.2020.607161

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 genes (named - ) 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 genes ( , and , 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 genes. The successful editing of the , and loci of 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 proved the absence of detectable non-specific mutations. The induced mutations were stably inherited in the T and T 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.