| Autor: |
Giulia Vittoria Ruta, Matteo Ciciani, Eyemen Kheir, Michele Domenico Gentile, Simone Amistadi, Antonio Casini, Anna Cereseto |
| Jazyk: |
angličtina |
| Rok vydání: |
2024 |
| Předmět: |
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| Zdroj: |
Genome Biology, Vol 25, Iss 1, Pp 1-15 (2024) |
| Druh dokumentu: |
article |
| ISSN: |
1474-760X |
| DOI: |
10.1186/s13059-024-03215-9 |
| Popis: |
Abstract Background 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. Results Here, we develop 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 is validated with CjCas9, generating an enhanced variant, UltraCjCas9, following directed evolution rounds. UltraCjCas9 is up to 12-fold more active in mammalian endogenous genomic loci, while preserving high genome-wide specificity. Conclusions We report a eukaryotic pipeline allowing enhancement of Cas9 systems, setting the ground to unlock the multitude of RNA-guided nucleases existing in nature. |
| Databáze: |
Directory of Open Access Journals |
| Externí odkaz: |
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