Enhancing prime editor activity by directed protein evolution in yeast

Yanik Weber, Desirée Böck, Anastasia Ivașcu, Nicolas Mathis, Tanja Rothgangl, Eleonora I. Ioannidi, Alex C. Blaudt, Lisa Tidecks, Máté Vadovics, Hiromi Muramatsu, Andreas Reichmuth, Kim F. Marquart, Lucas Kissling, Norbert Pardi, Martin Jinek and Gerald Schwank ()
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Yanik Weber: University of Zurich
Desirée Böck: University of Zurich
Anastasia Ivașcu: University of Zurich
Nicolas Mathis: University of Zurich
Tanja Rothgangl: University of Zurich
Eleonora I. Ioannidi: University of Zurich
Alex C. Blaudt: University of Zurich
Lisa Tidecks: University of Zurich
Máté Vadovics: University of Pennsylvania
Hiromi Muramatsu: University of Pennsylvania
Andreas Reichmuth: University of Zurich
Kim F. Marquart: University of Zurich
Lucas Kissling: University of Zurich
Norbert Pardi: University of Pennsylvania
Martin Jinek: University of Zurich
Gerald Schwank: University of Zurich

Nature Communications, 2024, vol. 15, issue 1, 1-11

Abstract: Abstract Prime editing is a highly versatile genome editing technology that enables the introduction of base substitutions, insertions, and deletions. However, compared to traditional Cas9 nucleases prime editors (PEs) are less active. In this study we use OrthoRep, a yeast-based platform for directed protein evolution, to enhance the editing efficiency of PEs. After several rounds of evolution with increased selection pressure, we identify multiple mutations that have a positive effect on PE activity in yeast cells and in biochemical assays. Combining the two most effective mutations – the A259D amino acid substitution in nCas9 and the K445T substitution in M-MLV RT – results in the variant PE_Y18. Delivery of PE_Y18, encoded on DNA, mRNA or as a ribonucleoprotein complex into mammalian cell lines increases editing rates up to 3.5-fold compared to PEmax. In addition, PE_Y18 supports higher prime editing rates when delivered in vivo into the liver or brain. Our study demonstrates proof-of-concept for the application of OrthoRep to optimize genome editing tools in eukaryotic cells.

Date: 2024
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DOI: 10.1038/s41467-024-46107-z

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