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Enhancing prime editor activity by directed protein evolution in yeast

Author

Listed:
  • 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
    Institute of Molecular Health Sciences, ETH Zurich)

  • Lucas Kissling

    (University of Zurich)

  • Norbert Pardi

    (University of Pennsylvania)

  • Martin Jinek

    (University of Zurich)

  • Gerald Schwank

    (University of Zurich)

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.

Suggested Citation

  • 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. , 2024. "Enhancing prime editor activity by directed protein evolution in yeast," Nature Communications, Nature, vol. 15(1), pages 1-11, December.
    • Handle: RePEc:nat:natcom:v:15:y:2024:i:1:d:10.1038_s41467-024-46107-z
    DOI: 10.1038/s41467-024-46107-z
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    References listed on IDEAS

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    1. Zhenxing Yu & Zhike Lu & Jingjing Li & Yingying Wang & Panfeng Wu & Yini Li & Yangfan Zhou & Bailun Li & Heng Zhang & Yingzheng Liu & Lijia Ma, 2022. "PEAC-seq adopts Prime Editor to detect CRISPR off-target and DNA translocation," Nature Communications, Nature, vol. 13(1), pages 1-13, December.
    2. Kevin M. Esvelt & Jacob C. Carlson & David R. Liu, 2011. "A system for the continuous directed evolution of biomolecules," Nature, Nature, vol. 472(7344), pages 499-503, April.
    3. Paul Jannis Zurek & Philipp Knyphausen & Katharina Neufeld & Ahir Pushpanath & Florian Hollfelder, 2020. "UMI-linked consensus sequencing enables phylogenetic analysis of directed evolution," Nature Communications, Nature, vol. 11(1), pages 1-10, December.
    4. Andrew V. Anzalone & Peyton B. Randolph & Jessie R. Davis & Alexander A. Sousa & Luke W. Koblan & Jonathan M. Levy & Peter J. Chen & Christopher Wilson & Gregory A. Newby & Aditya Raguram & David R. L, 2019. "Search-and-replace genome editing without double-strand breaks or donor DNA," Nature, Nature, vol. 576(7785), pages 149-157, December.
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