Engineering dynamic cell cycle control with synthetic small molecule-responsive RNA devices

Kathy Y. Wei, Christina D. Smolke

The cell cycle plays a key role in human health and disease, including development and cancer. The ability to easily and reversibly control the mammalian cell cycle could mean improved cellular reprogramming, better tools for studying cancer, more efficient gene therapy, and improved heterologous protein production for medical or industrial applications. We engineered RNA-based control devices to provide specific and modular control of gene expression in response to exogenous inputs in living cells. Specifically, we identified key regulatory nodes that arrest U2-OS cells in the G0/1 or G2/M phases of the cycle. We then optimized the most promising key regulators and showed that, when these optimized regulators are placed under the control of a ribozyme switch, we can inducibly and reversibly arrest up to ~80 % of a cellular population in a chosen phase of the cell cycle. Characterization of the reliability of the final cell cycle controllers revealed that the G0/1 control device functions reproducibly over multiple experiments over several weeks. To our knowledge, this is the first time synthetic RNA devices have been used to control the mammalian cell cycle. This RNA platform represents a general class of synthetic biology tools for modular, dynamic, and multi-output control over mammalian cells.