(4aj) Dynamically Reshaping Signaling Networks to Program Cell Fate Via Genetic Controllers

Engineering of cell fate through synthetic gene circuits requires methods to precisely implement control around native decision-making pathways, and offers the potential to direct developmental programs and intervene in aberrantly activated cell processes. We demonstrate a class of genetic control systems, molecular network diverters, that interface with a native signaling pathway to route cells to divergent fates in response to environmental signals without modification of native genetic material. A readily translatable method for identifying control points within natural networks is described that enables the construction of synthetic control systems that activate or attenuate native pathways to direct cell fate. We integrate opposing genetic programs by developing network architectures for reduced antagonism and demonstrate rational tuning of performance. The integrated, optimized system allowed us to selectively activate and attenuate signaling through a native MAPK pathway to conditionally redirect, or route, cells to one of three distinct fates. Additionally, we constructed a model to guide the design of similar control systems, highlighting critical parameters for balancing the opposing diverter functions. The extension of these control strategies to mammalian systems should facilitate the engineering of complex cellular signaling systems.