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bioRxiv · 10.1101/2023.09.18.558227

Eukaryotic-driven directed evolution of Cas9 nucleases

Ruta, G. V.·Ciciani, M.·Kheir, E.·Gentile, M. D.·Amistadi, S.·Casini, A.·Cereseto, A.
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Abstract

Full exploitation of the natural reservoir of CRISPR-Cas nucleases from prokaryotes for genome editing is limited by the suboptimal activity of these enzymes in mammalian cells. 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 a poorly efficient Cas9 nuclease from Campylobacter jejuni, 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. Here we report a eukaryotic pipeline allowing enhancement of Cas9 systems, setting the ground to unlock the multitude of RNA-guided nucleases existing in nature.

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Ruta, G. V., Ciciani, M., Kheir, E., Gentile, M. D., Amistadi, S., Casini, A., Cereseto, A.. 2023-09-18. Eukaryotic-driven directed evolution of Cas9 nucleases. https://doi.org/10.1101/2023.09.18.558227

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