Composing Mammalian Gene Expression Programs Using the Comet Synthetic Transcription Control Ensemble

Mammalian cells can be programmed for numerous applications, ranging from customized cell-based therapeutics to tools for probing fundamental biological questions. Towards these goals, we recently developed the Composable Mammalian Elements of Transcription (COMET) tootlkit. COMET comprises Zinc-finger (ZF)-based synthetic transcription factors (sTFs), including both activating (ZFa) and inhibitory (ZFi) proteins, and engineered promoters that are responsive to these sTFs. This library of orthogonal sTFs and promoters, with tunable gene regulation properties, enables the composition of sophisticated cellular programs. We report here on the characterization of COMET, as well as two distinct initial applications: performing logical evaluation and implementing a gene circuit with customized dynamics. Plasmids encoding sTFs or biosensors and cognate promoter-reporter constructs were transfected into HEK293FT cells, which were analyzed via flow cytometry.

Parameters for designing sTF-responsive, engineered promoters were elucidated, and notably, ZFa-induced gene expression spanned three orders of magnitude. Removing the activation domain from the ZFa created sTFs that could inhibit these promoters (ZFi), and addition of a domain that enhances steric occlusion to the ZFi strengthened this inhibition. Furthermore, ZFa that activate transcription only in the presence of a small molecule ligand were constructed by splitting the ZF from the activation domain and fusing each to a rapamycin-inducible dimerization domain to create RaZF. RaZF were able to activate gene expression only in the presence of rapamycin. Several activation domains were tested and shown to confer various levels of induction. To demonstrate the ability of COMET to integrate multiple signals, promoters that required two and three ZFa to robustly activate gene expression were designed and tested, enabling cells to perform logical evaluation of multiple cues. Finally, COMET sTFs and promoters were used to design several dynamic circuits providing temporal control of gene expression. By employing positive and negative feedback, sequential activation and pulse circuits can drive gene expression in a programmable fashion.

We are currently leveraging this new biochemical tool set for applications including programmable engineered cell-based therapies and customizable gene expression programs to interrogate and drive stem cell differentiation. The COMET toolkit will be of substantial use to both fundamental biologists and the mammalian synthetic biology community, by expanding our capabilities to program cells to implement a wide variety of desired behaviors.