| Online-Ressource |
Verfasst von: | Stadelmann, Tobias [VerfasserIn]  |
| Heid, Daniel [VerfasserIn]  |
| Jendrusch, Michael [VerfasserIn]  |
| Mathony, Jan [VerfasserIn]  |
| Aschenbrenner, Sabine [VerfasserIn]  |
| Rosset, Stéphane [VerfasserIn]  |
| Correia, Bruno E. [VerfasserIn]  |
| Niopek, Dominik [VerfasserIn]  |
Titel: | A deep mutational scanning platform to characterize the fitness landscape of anti-CRISPR proteins |
Verf.angabe: | Tobias Stadelmann, Daniel Heid, Michael Jendrusch, Jan Mathony, Sabine Aschenbrenner, Stéphane Rosset, Bruno E. Correia and Dominik Niopek |
E-Jahr: | 2024 |
Jahr: | 11 December 2024 |
Umfang: | 14 S. |
Illustrationen: | Illustrationen |
Fussnoten: | Online verfügbar: 18. November 2024 ; Gesehen am 21.05.2025 |
Titel Quelle: | Enthalten in: Nucleic acids research |
Ort Quelle: | Oxford : Oxford Univ. Press, 1974 |
Jahr Quelle: | 2024 |
Band/Heft Quelle: | 52(2024), 22, Artikel-ID e103, Seite 1-14 |
ISSN Quelle: | 1362-4962 |
Abstract: | Deep mutational scanning is a powerful method for exploring the mutational fitness landscape of proteins. Its adaptation to anti-CRISPR proteins, which are natural CRISPR-Cas inhibitors and key players in the co-evolution of microbes and phages, facilitates their characterization and optimization. Here, we developed a robust anti-CRISPR deep mutational scanning pipeline in Escherichia coli that combines synthetic gene circuits based on CRISPR interference with flow cytometry coupled sequencing and mathematical modeling. Using this pipeline, we characterized comprehensive single point mutation libraries for AcrIIA4 and AcrIIA5, two potent inhibitors of CRISPR-Cas9. The resulting mutational fitness landscapes revealed considerable mutational tolerance for both Acrs, suggesting an intrinsic redundancy with respect to Cas9 inhibitory features, and - for AcrIIA5 - indicated mutations that boost Cas9 inhibition. Subsequent in vitro characterization suggested that the observed differences in inhibitory potency between mutant inhibitors were mostly due to changes in binding affinity rather than protein expression levels. Finally, to demonstrate that our pipeline can inform Acrs-based genome editing applications, we employed a selected subset of mutant inhibitors to increase CRISPR-Cas9 target specificity by modulating Cas9 activity. Taken together, our work establishes deep mutational scanning as a powerful method for anti-CRISPR protein characterization and optimization. |
DOI: | doi:10.1093/nar/gkae1052 |
URL: | Bitte beachten Sie: Dies ist ein Bibliographieeintrag. Ein Volltextzugriff für Mitglieder der Universität besteht hier nur, falls für die entsprechende Zeitschrift/den entsprechenden Sammelband ein Abonnement besteht oder es sich um einen OpenAccess-Titel handelt.
kostenfrei: Volltext: https://doi.org/10.1093/nar/gkae1052 |
| kostenfrei: Volltext: https://academic.oup.com/nar/article/52/22/e103/7903370?lo |
| DOI: https://doi.org/10.1093/nar/gkae1052 |
Datenträger: | Online-Ressource |
Sprache: | eng |
K10plus-PPN: | 1926249372 |
Verknüpfungen: | → Zeitschrift |
¬A¬ deep mutational scanning platform to characterize the fitness landscape of anti-CRISPR proteins / Stadelmann, Tobias [VerfasserIn]; 11 December 2024 (Online-Ressource)