Mathony, JanHarteveld, ZanderSchmelas, Carolinzu Belzen, Julius UpmeierAschenbrenner, SabineSun, WeiHoffmann, Mareike D.Stengl, ChristinaScheck, AndreasGeorgeon, SandrineRosset, StephaneWang, YanliGrimm, DirkEils, RolandCorreia, Bruno E.Niopek, Dominik2020-04-302020-04-302020-04-302020-04-1310.1038/s41589-020-0518-9https://infoscience.epfl.ch/handle/20.500.14299/168497WOS:000526289200001Anti-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.Biochemistry & Molecular BiologyBiochemistry & Molecular Biologygenomealgorithmtext::journal::journal article::research article