This project aims to develop and implement effective methods of cutting-edge CRISPR/Cas genome editing for the identification of individual genes underlying quantitative trait loci (QTLs) for agronomically important crop traits. QTL intervals can contain hundreds of genes, and narrowing down the number of candidate genes, eventually identifying the QTL's causal gene, is a common approach in applied and fundamental research. Transferring a QTL’s causal variant gene with as few as possible other genes into elite breeding varieties requires time and effort, which this project aims to decrease. The societal issue and benefit that is addressed is the speed at which new crop varieties can be bred for facing challenges concerning developing a more sustainable agriculture. In this project, the newly developed techniques will be applied to two QTLs for tomato fruit firmness originating from a preceding TKI project and relevant for reducing postharvest food waste. The speed and efficacy of the new techniques will be compared with those of the classical approach of crossing and backcrossing for narrowing down the QTL interval to a few or, eventually, a single causal gene variant. The deliverables of the project will be a set of approaches for knocking out multiple genes individually or in combinations, as well as for deleting multiple adjacent genes. Further application to the two firmness QTLs will deliver gene-edited tomato lines and a set of recombinants, eventually leading to the underlying causal genes. These lines will be thoroughly characterized for firmness and additional phenotypes. Finally, the participating breeding companies will introduce the relevant gene variants by crossing or mutagenesis in their own elite material.