Malus genome editing via CRISPR/Cas9 to develop sustainable pest free apples

ended

Apple is one of the most important crop. Pathogens causing powdery mildew, fire blight, and scab represent serious threats to apple cultivation. These pathogens are mainly controlled by pesticides with a negative impact on the environment and possibly on the health of the consumers on the long term. Resistant cultivars are the most effective and sustainable means to control these threats. Resistance to pathogens is usually conferred to apples by the introgression of R-genes from wild cultivars but such approach is not durable. Suppression of genes used by pathogens for infection (S-genes) can reduce pathogen attack. CRISPR/Cas9-mediated genome editing was used to mutate S-genes but these attempts were limited to single genes and were based on the stable integration of the editing machinery preventing these plants from being accepted by customers. The main objective of this project is to use CRISPR/Cas9 to create a multi-resistant apple variety by the simultaneous mutation of S-genes which are responsible for susceptibility to powdery mildew and fire blight. The main challenge will be to introduce such mutations using a “transgene-free” approach which is not based on protoplast transformation. The innovative approach consists on delivering the editing machinery via Agrobacterium tumefaciens or particle bombardment to apple cells and then eliminate editing components after mutation using an inducible site-specific recombination-based system. The final goal is to obtain a multi-resistant, sustainable and transgene-free apple. The second objective of the project is to develop a method for regeneration of apple plants from protoplast which safeguard genetic stability during regeneration to allow genome editing in apple protoplasts using pre-assembled Cas9-sgRNA ribonucleoproteins.