Nature Chemical Biology, 13 April, 2020, DOI：https://doi.org/10.1038/s41589-020-0518-9
Computational design of anti-CRISPR proteins with improved inhibition potency
Jan Mathony, Zander Harteveld, Carolin Schmelas, Julius Upmeier zu Belzen, Sabine Aschenbrenner, Wei Sun, Mareike D. Hoffmann, Christina Stengl, Andreas Scheck, Sandrine Georgeon, Stéphane Rosset, Yanli Wang, Dirk Grimm, Roland Eils, Bruno E. Correia & Dominik Niopek
Anti-CRISPR (Acr) proteins are powerful tools to control CRISPR–Cas technologies. However, the available Acr repertoire is limited to naturally occurring variants. Here, we applied structure-based design on AcrIIC1, a broad-spectrum CRISPR–Cas9 inhibitor, to improve its efficacy on different targets. We first show that inserting exogenous protein domains into a selected AcrIIC1 surface site dramatically enhances inhibition of Neisseria meningitidis (Nme)Cas9. Then, applying structure-guided design to the Cas9-binding surface, we converted AcrIIC1 into AcrIIC1X, a potent inhibitor of the Staphylococcus aureus (Sau)Cas9, an orthologue widely applied for in vivo genome editing. Finally, to demonstrate the utility of AcrIIC1X for genome engineering applications, we implemented a hepatocyte-specific SauCas9 ON-switch by placing AcrIIC1X expression under regulation of microRNA-122. Our work introduces designer Acrs as important biotechnological tools and provides an innovative strategy to safeguard CRISPR technologies.