S. Schürle and A. Schubert will use genome engineering with the aim to provide mechanistic insights into signalling pathways in the tumour–microbe–drug interplay. For this, they will utilise and optimise existing organ-on-a-chip models of CRC that mimic physiologically relevant biochemical and physical cues of tumour and metastatic niches. The system provides means to perform multiparametric analyses of bacterial colonisation dynamics including tumour response to drug perturbations using advanced and multiscale imaging techniques. They will employ CRISPR/Cas9-mediated genome engineering including fluorescent labelling of signalling mediators to mechanistically characterise Wnt signalling activation. Combining organ-on-a-chip models with confocal fluorescence microscopy will then enable live monitoring of signalling activation in response to stimulation with tumour-resident and therapeutic bacteria, as well as to treatment with established chemotherapy regimens and novel drug combinations from other sub-projects. Building on the gained mechanistic insights, they will design and assess therapeutic bacterial vectors for Wnt-targeted CRC therapy in vitro and in vivo.