Eukaryotic-driven directed evolution of Cas9 nucleases

Further advancement of genome editing highly depends on the development of tools with higher compatibility with eukaryotes. A multitude of described Cas9s have great potential but require optimization for genome editing purposes. Among these, the Cas9 from Campylobacter jejuni, CjCas9, has a favorable small size, facilitating delivery in mammalian cells. Nonetheless, its full exploitation is limited by its poor editing activity. Here we developed a Eukaryotic Platform to Improve Cas Activity (EPICA) to steer weakly active Cas9 nucleases into highly active enzymes by directed evolution. The EPICA platform is obtained by coupling Cas nuclease activity with yeast auxotrophic selection followed by mammalian cell selection through a sensitive reporter system. EPICA was validated with CjCas9, generating an enhanced variant, UltraCjCas9, following directed evolution rounds. UltraCjCas9 was up to 12-fold more active in mammalian endogenous genomic loci, while preserving high genome-wide specificity.