GE-Reduced browning banana was developed using the CRISPR/Cas9 technology delivered through Agrobacterium-mediated transformation into embryogenic cells of Banana cultivar Grande Naine.
The intended phenotype of GE-Reduced browning bananas is decreased levels of polyphenol oxidase proteins in order to reduce banana fruit browning. The CRISPR/Cas9 system with two specific gRNAs was used to introduce a targeted double-strand break (DSB) within the sequence of a PPO gene. The induced DSB in one allele of the target gene is causing a frameshift mutation in the coding sequence, preventing the functional protein from being produced. No external repair template was provided.
It is unclear, which PPO gene was targeted in the final GE-Reduced browning banana plant. The genome of banana contains 10 endogenous PPO genes. PPO1 is the highest-expressed PPO gene in the fruit tissue, and during fruit ripening PPO1 and PPO6 proteins are most abundant (Qin et al., 2023).
In the Patent WO 2023/275255 from Tropic Biosciences, a more detailed analysis of a PPO1-modified plant line is described. Therefore, PPO1 might be the targeted gene in the final plant line.
Molecular characterization using quantitative PCR and sequencing confirmed that the final selected plant does not contain any introduced DNA from the CRISPR/Cas9 system. The application document states that the genome of the final plant will be analyzed by whole-genome sequencing to confirm the presence of the intended modification and absence of modifications in potential secondary targets or any unintentionally integrated DNA.
Sources:
- USDA-APHIS confirmation request 21-356-01cr
- Patent WO 2023/275255
- Qin et al. (2023), Genome-wide analysis of the polyphenol oxidase gene family reveals that MaPPO1 and MaPPO6 are the main contributors to fruit browning in Musa acuminate. Frontiers in Plant Science 14:1125375