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Abstract

An increasingly powerful set of new CRISPR/Cas-based methods is becoming available for directed evolution of proteins in mammalian cells. Although in vitro techniques or microbial expression systems have been dominating directed evolution, there are now promising approaches to diversify proteins in mammalian cells in situ. This can be achieved by simple indel mutagenesis or more sophisticated homology repair mechanisms for cassette mutagenesis of coding sequences. Cas9 variant fusions to base editors and other effectors pose another promising way to introduce diversity into proteins. CRISPR/Cas9-based directed evolution in mammalian cells opens a new exciting era of discovery for the many classes of proteins for which a mammalian cellular context is preferable.