<b>Capsid-complemented Adaptation of Engineered Sindbis virus with Activity-gated RNA-knockdown</b>

Directed evolution is a powerful approach for enhancing biomolecule functionality by iteratively selecting and enriching fitter genotypes. Evolving mammalian protein targets in lower organisms is confounded by differences in post-translational modification. Here, we describe a mammalian platform for <i>in vivo </i>hypermutation with manual screening of variants. Capsid-complemented Adaptation of Engineered Sindbis virus with Activity-gated RNA-knockdown (CAESAR), leverages an engineered Sindbis virus variant that enables coupling of mutagenesis and selection processes. We demonstrate the reproducibility and efficacy of CAESAR through multiple campaigns, showcasing its ability to generate solutions for the evolution of tTA using an RNA-based targeting system. CAESAR addresses the limitations of previous platforms, allowing for serial passaging and transgene maintenance with a selection circuit that enables gene flow and mutagenesis between rounds through the use of defective interfering particles and defective helper RNA. This three-component, RNA-based platform opens new avenues for mammalian hypermutation systems, enabling the creation of bespoke genes with improved functionality