Improving the beneficial effects of tomato on human health by reducing the levels of allergenic molecules

Solanum lycopersicum (also known as tomato) is one of the most cultivated fruit crops all over the world. Nowadays, its importance is due to the high content of bioactive compounds beneficial to human health. However, tomatoes are also known for containing anti-nutritional compounds and allergenic proteins. Anti-nutritional compounds, like glycoalkaloids, are toxic biomolecules for human health. Instead, allergenic proteins such as Sola I 1-7, Thaumatin-like proteins, and polygalacturonase 2a can induce allergic reactions. To date, 26 proteins, including isoforms, from tomatoes have been reported as potential allergens. Tomato allergens are listed in the WHO/IUIS (http://www.allergen.org) and Allergome (http://www.allergome.org) databases.

To this end, it is crucial to reduce the number of allergens in tomatoes to improve their qualitative aspects, as well as their beneficial properties.

The research will involve different activities as reported below:

• The genetic background of the editing experiments will be the Moneymaker cultivar, where the CRISPR/Cas9 genome editing will be applied to knockout two allergenic proteins: thaumatin-like proteins (TLPs), belonging to the PR-5 family, with the PR-NP24 protein expressed in the tomato exocarp; and polygalacturonase 2a (PG2a), an enzyme involved in the degradation of pectin. 
• Different gRNAs for the knockout of these genes will be designed, and then the DNA construct will be assembled using Goldenbraid. The tomato transformations using Agrobacterium tumefaciens will be performed. 
• The fruits of plants edited with the homozygous loss-of-function mutations (F2) will be analyzed for the effective loss of allergenicity.
• Eventually our approach will allow us to obtain tomato lines with low or no allergenic proteins to improve the beneficial effects of tomatoes. In addition, our results will show that it is possible to target and stack different genes using CRISPR/Cas9 genome editing. Finally, breath tests will be performed on patients with and without tomato allergies to demonstrate low allergenicity.

• The main result is to obtain tomato lines with low or no allergenic proteins to improve the beneficial effects of tomatoes. 
• The additional result is the demonstration that it is possible to target and stack different genes using CRISPR/Cas9 genome editing.