Integrative Approaches to Synthetic Biology in Human Cells

Abstract

Synthetic biology endeavors to develop systems and processes to aid in the rapid, predictable, and reproducible design of biological systems not only for use as research tools and also but to address unmet needs in industry and medicine. Borrowing heavily from other engineering disciplines, synthetic biologists employ a variety of approaches in rational, computational, and adaptive design to tackle a myriad of challenges with biology-based solutions. In this dissertation, we combine these different frameworks in the development of novel tools to explore transcriptional perturbation, cytokine signaling dynamics, and heterogeneous response in human cells. In Chapter 2, we repurpose CRISPR/Cas9 as a modular platform for simultaneous differential transcriptional regulation of multiple genes. In Chapters 3 and 4, we explore design principles for engineering minimal sensors of interferon signaling and for enhancing their signal and dynamic range, enabling investigation of the spatio-temporal dynamics for the innate antiviral response. Finally, in Chapter 5, we use CRISPR/Cas9 to engineer transcriptional positive feedback, creating a circuit that can record transient exposure to an environmental stimulus while also exploring challenges associated with stable integration of heterologous circuits in human cells. Importantly, the principles uncovered here in designing, implementing, and evaluating these synthetic devices are generalizable and offer key insights into the development and optimization of other sensors and circuits.