Coupling Cas9 to artificial inhibitory domains enhances CRISPR-Cas9 target specificity
The limited target specificity of CRISPR-Cas nucleases poses a challenge with respect to their application in research and therapy. Here, we present a simple and original strategy to enhance the specificity of CRISPR-Cas9 genome editing by coupling Cas9 to artificial inhibitory domains. Applying a c...
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| Hauptverfasser: | , , , , , |
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| Dokumenttyp: | Article (Journal) |
| Sprache: | Englisch |
| Veröffentlicht: |
05 Feb 2020
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| In: |
Science advances
Year: 2020, Jahrgang: 6, Heft: 6 |
| ISSN: | 2375-2548 |
| DOI: | 10.1126/sciadv.aay0187 |
| Online-Zugang: | Verlag, lizenzpflichtig, Volltext: https://doi.org/10.1126/sciadv.aay0187 Verlag, lizenzpflichtig, Volltext: https://advances.sciencemag.org/content/6/6/eaay0187 
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| Verfasserangaben: | Sabine Aschenbrenner, Stefan M. Kallenberger, Mareike D. Hoffmann, Adrian Huck, Roland Eils, Dominik Niopek |
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| 520 | |a The limited target specificity of CRISPR-Cas nucleases poses a challenge with respect to their application in research and therapy. Here, we present a simple and original strategy to enhance the specificity of CRISPR-Cas9 genome editing by coupling Cas9 to artificial inhibitory domains. Applying a combination of mathematical modeling and experiments, we first determined how CRISPR-Cas9 activity profiles relate to Cas9 specificity. We then used artificially weakened anti-CRISPR (Acr) proteins either coexpressed with or directly fused to Cas9 to fine-tune its activity toward selected levels, thereby achieving an effective kinetic insulation of ON- and OFF-target editing events. We demonstrate highly specific genome editing in mammalian cells using diverse single-guide RNAs prone to potent OFF-targeting. Last, we show that our strategy is compatible with different modes of delivery, including transient transfection and adeno-associated viral vectors. Together, we provide a highly versatile approach to reduce CRISPR-Cas OFF-target effects via kinetic insulation. - Fusing attenuated anti-CRISPR proteins to Cas9 improves specificity via kinetic insulation of ON- and OFF-target editing events. - Fusing attenuated anti-CRISPR proteins to Cas9 improves specificity via kinetic insulation of ON- and OFF-target editing events. | ||
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