Caliciviral protein-based artificial translational activator for mammalian gene circuits with RNA-only delivery

Synthetic RNA-based gene circuits enable sophisticated gene regulation without the risk of insertional mutagenesis. While various RNA binding proteins have been used for translational repression in gene circuits, the direct translational activation of synthetic mRNAs has not been achieved. Here we develop Caliciviral VPg-based Translational activator (CaVT), which activates the translation of synthetic mRNAs without the canonical 5'-cap. The level of translation can be modulated by changing the locations, sequences, and modified nucleosides of CaVT-binding motifs in the target mRNAs, enabling the simultaneous translational activation and repression of different mRNAs with RNA-only delivery. We demonstrate the efficient regulation of apoptosis and genome editing by tuning translation levels with CaVT. In addition, we design programmable CaVT that responds to endogenous microRNAs or small molecules, achieving both cell-state-specific and conditional translational activation from synthetic mRNAs. CaVT will become an important tool in synthetic biology for both biological studies and future therapeutic applications.