Enhancing prime editor activity by directed protein evolution in yeast

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 protei...

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Vydáno v:Nature communications Ročník 15; číslo 1; s. 2092 - 11
Hlavní autoři: Weber, Yanik, Böck, Desirée, Ivașcu, Anastasia, Mathis, Nicolas, Rothgangl, Tanja, Ioannidi, Eleonora I., Blaudt, Alex C., Tidecks, Lisa, Vadovics, Máté, Muramatsu, Hiromi, Reichmuth, Andreas, Marquart, Kim F., Kissling, Lucas, Pardi, Norbert, Jinek, Martin, Schwank, Gerald
Médium: Journal Article
Jazyk:angličtina
Vydáno: London Nature Publishing Group UK 07.03.2024
Nature Publishing Group
Nature Portfolio
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Amino Acid Substitution
Amino acids
Animals
Biological Assay
Brain
Cell Line
Cell lines
CRISPR-Cas Systems - genetics
Editing
Evolution
Genome editing
Genomes
Humanities and Social Sciences
In vivo methods and tests
Mammals
mRNA
multidisciplinary
Mutation
Nuclease
Proteins
Saccharomyces cerevisiae - genetics
Science
Science (multidisciplinary)
Substitutes
Yeast
ISSN:2041-1723, 2041-1723
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Popis
Shrnutí: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. Compared to traditional Cas9 nucleases prime editors (PEs) are less active. Here the authors use OrthoRep, a yeast-based platform for directed protein evolution to enhance the editing efficiency of PEs: they identify mutations that have a positive effect on kinetics and use this knowledge to generate an efficient in vivo PE.
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ISSN:2041-1723
2041-1723
DOI:10.1038/s41467-024-46107-z