Engineered Protease-Responsive RNA-Binding Proteins (RBPs) to Expand the Toolbox of Synthetic Circuits in Mammalian Cells

Genetically encoded sensor–actuator circuits aim at reprogramming cellular functions and are inspired by intracellular networks: from the input signal (sensor) to the desired output response (actuator). In the last years, circuits with posttranscriptional regulation of gene expression have aroused great interest for their potential in the biomedical space. Posttranscriptional modulation can be achieved with ribozymes, riboswitches (simple regulatory elements based on RNA secondary structures), noncoding RNAs, and RNA-binding proteins (RBPs). RBPs are proteins that recognize specific motifs on the mRNA target inducing mRNA decay or translation inhibition. The use of RBPs deriving from different species in mammalian cells has allowed to create sophisticated and multilayered regulatory networks, addressing the previous limitation of regulatory orthogonal parts that can be assembled in synthetic devices. In this chapter, we describe the engineering and tests of protease-responsive RNA-binding proteins (L7Ae and MS2-cNOT7) to expand the toolbox of synthetic circuits in mammalian cells.