Comet: A Novel Transcription Toolbox for Engineering Cellular Therapies

Engineering mammalian cells to serve as therapeutic or diagnostic devices relies heavily upon technologies for controlling gene expression. In many applications, signals received by receptors or other biosensors are ultimately transduced into a gene expression event, such as the production of a therapeutic protein or diagnostic readout, through a pathway comprising one or more transcription factors. Customized gene expression programs, including those developed using the approaches of mammalian synthetic biology, are currently limited to using either native transcription factors, which are subject to cross-regulation and interference with native cellular functions, or to using a small set of engineered transcriptional regulators, such as tTA and Gal4. To realize the goal of building sophisticated cellular programs, a larger set of orthogonal, well-characterized transcriptional regulators is needed. Towards this goal, we have developed and characterized the Composable Mammalian Elements of Transcription (COMET)—an ensemble of transcription factors and cognate promoters that enable the design and tuning of mammalian genetic circuits to an extent not previously possible. COMET currently comprises 53 activating and 38 inhibitory zinc-finger-based regulators, built using modular domains, and 96 cognate promoters; this system also enables the rapid construction of additional parts as needed for specific applications. The high specificity of zinc-finger DNA binding domains makes these transcription factors highly orthogonal. These parts were systematically characterized to generate a quantitative map of gene regulation properties. We demonstrated the utility of COMET by modulating gene expression over three orders of magnitude, introducing chemically inducible control, and by implementing single-layer, design-driven Boolean logic. Furthermore, we developed a mathematical model that accurately explains these behaviors and can be applied to guide the design of new circuits. Altogether, COMET is a powerful tool that enables bioengineers to rapidly design and implement custom genetic programs in mammalian cells.