PROJECT SUMMARY Engineering non-proliferating-but-active (NPBA) probiotic for breast-cancer gene therapy A major breast-cancer treatment challenge is the debilitating effects of systemic therapies. To create more tolerable and effective therapies against breast cancer, considerable effort is being directed to using CRISPR- Cas to target the molecular origins of the disease. However, current methods face multiple challenges in delivering CRISPR-Cas to breast cancer cells, including low penetration of solid tumors, low delivery amount, and small size of CRISPR-Cas. To address the challenges, we will engineer a new kind of CRISPR-Cas delivery vehicle for breast cancer cells using non-proliferating-but-active (NPBA) probiotic. Our preliminary work demonstrates that the NPBA probiotic conduct metabolism, move in aqueous environments, and does not proliferate. We ultimately envisage that the NPBA probiotic can be introduced into the breast ducts or tumors to migrate towards cancer cells and then deliver a CRISPR-Cas construct that targets specific oncogenic mechanisms. Hence, this proposal aims to engineer these NPBA probiotic to deliver genes or proteins to breast cancer cells. Our team will leverage expertise in gene expression control, bacterial information processing, molecular tools, and breast cancer models. We will pursue two parallel aims that correspond to the engineering and testing of the NPBA Escherichia coli Nissle 1917. 1) Enable NPBA probiotics to invade mammalian cells before delivering CRISPR-Cas. This aim will establish the basic CRISPR-Cas-delivery function of the NPBA probiotics. 2) Enhance the invasion and CRISPR-Cas delivery efficacy of the NPBA probiotic in silencing the ErbB2 oncogene of breast cancer cells. This project will create a new paradigm that uses non-proliferating-but- active probiotics for effective and specific delivery of CRISPR-Cas to cancer cells. Our work will also form the foundation for using the NPBA probiotics to target various oncogenic and proliferative pathways of cancer cells. Future work may also investigate any dispersion of the NPBA probiotics into the surrounding tissues and throughout the host.