Coupling Cas9 to artificial inhibitory domains enhances CRISPR-Cas9 target specificity

• Verfasst von: Aschenbrenner, Sabine [VerfasserIn]
• Verfasst von: Kallenberger, Stefan M. [VerfasserIn]
• Verfasst von: Hoffmann, Mareike [VerfasserIn]
• Verfasst von: Huck, Adrian [VerfasserIn]
• Verfasst von: Eils, Roland [VerfasserIn]
• Verfasst von: Niopek, Dominik [VerfasserIn]
• Titel: Coupling Cas9 to artificial inhibitory domains enhances CRISPR-Cas9 target specificity
• Verf.angabe: Sabine Aschenbrenner, Stefan M. Kallenberger, Mareike D. Hoffmann, Adrian Huck, Roland Eils, Dominik Niopek
• E-Jahr: 2020
• Jahr: 05 Feb 2020
• Umfang: 12 S.
• Fussnoten: Gesehen am 30.03.2020
• Titel Quelle: Enthalten in: Science advances
• Ort Quelle: Washington, DC [u.a.] : Assoc., 2015
• Jahr Quelle: 2020
• Band/Heft Quelle: 6(2020,6) Artikel-Nummer eaay0187, Seite 1-12
• ISSN Quelle: 2375-2548
• DOI: doi:10.1126/sciadv.aay0187
• Datenträger: Online-Ressource
• Sprache: eng
• K10plus-PPN: 1693503891

Abstract

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.