Abstract
Directed evolution is a powerful approach to obtain genetically-encoded sought-for traits. Compared to the prolonged adaptation regimes to mutations occurring under natural selection, directed evolution unlocks rapid screening and selection of mutants with improved traits from vast mutated sequence spaces. Many systems have been developed to search variant landscapes based on ex vivo or in vivo mutagenesis, to identify and select new-to-nature and optimized properties in biomolecules. Yet, the majority of such systems rely on tedious iterations of library preparation, propagation, and selection steps. Furthermore, among the relatively few in vivo directed evolution systems developed to mitigate handling of repetitive ex vivo steps, directed evolution of DNA is the standard approach. Here, we present the protocol for designing the transfer of genetic information from evolving RNA donors to DNA in baker's yeast, using CRISPR- and RNA-assisted in vivo directed evolution (CRAIDE). We use mutant T7 RNA polymerase to introduce mutations in RNA donors, while incorporation into DNA is directed by CRISPR/Cas9. As such, CRAIDE offers an opportunity to study fundamental questions, such as RNA's contribution to the evolution of DNA-based life on Earth. Graphic abstract: CRISPR- and RNA-assisted in vivo directed evolution (CRAIDE).