Nickase fidelity drives EvolvR-mediated diversification in mammalian cells

In vivo genetic diversifiers have previously enabled efficient searches of genetic variant fitness landscapes for continuous directed evolution. However, genomic diversification modalities shown to work in mammalian cells exclusively rely on deaminases to generate transition mutations within target loci, forfeiting access to the majority of missense mutations. Here, we engineered CRISPR-guided error-prone DNA polymerases (EvolvR) to diversify all four nucleotides within mammalian genomic loci for the first time.