Programmable mammalian translational modulators by CRISPR-associated proteins

The scarcity of qualified RNA-binding proteins hinders the development of artificial translational regulators and synthetic gene circuits. Here, the authors repurposed CRISPR-Cas proteins as translational regulators to build synthetic circuits. Translational modulation based on RNA-binding proteins can be used to construct artificial gene circuits, but RNA-binding proteins capable of regulating translation efficiently and orthogonally remain scarce. Here we report CARTRIDGE (Cas-Responsive Translational Regulation Integratable into Diverse Gene control) to repurpose Cas proteins as translational modulators in mammalian cells. We demonstrate that a set of Cas proteins efficiently and orthogonally repress or activate the translation of designed mRNAs that contain a Cas-binding RNA motif in the 5'-UTR. By linking multiple Cas-mediated translational modulators, we designed and built artificial circuits like logic gates, cascades, and half-subtractor circuits. Moreover, we show that various CRISPR-related technologies like anti-CRISPR and split-Cas9 platforms could be similarly repurposed to control translation. Coupling Cas-mediated translational and transcriptional regulation enhanced the complexity of synthetic circuits built by only introducing a few additional elements. Collectively, CARTRIDGE has enormous potential as a versatile molecular toolkit for mammalian synthetic biology.

Automated summary

The scarcity of qualified RNA-binding proteins hinders the development of artificial translational regulators and synthetic gene circuits. Here, the authors repurposed CRISPR-Cas proteins as translational regulators to build synthetic circuits. CARTRIDGE (Cas-Responsive Translational Regulation Integratable into Diverse Gene control) is a versatile molecular toolkit for mammalian synthetic biology that uses Cas proteins as translational modulators in mammalian cells. By linking multiple Cas-mediated translational modulators, artificial circuits like logic gates, cascades, and half-subtractor circuits can be designed and built.