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Computational design of anti-CRISPR proteins with improved inhibition potency and expanded specificity

By Jan Mathony, Zander Harteveld, Carolin Schmelas, Julius Upmeier zu Belzen, Sabine Aschenbrenner, Mareike D Hoffmann, Christina Stengl, Andreas Scheck, St├ęphane Rosset, Dirk Grimm, Roland Eils, Bruno E Correia, Dominik Niopek

Posted 27 Jun 2019
bioRxiv DOI: 10.1101/685032

Anti-CRISPR (Acr) proteins are bacteriophage-derived antagonists of CRISPR-Cas systems. To date, Acrs were obtained either by mining sequence databanks or experimentally screening phage collections, both of which yield a limited repertoire of naturally occurring variants. Here, we applied structure-based engineering on AcrIIC1, a broad-spectrum inhibitor of type II-C CRISPR systems, to improve its efficacy and expand its specificity. We first show that fusing exogenous protein domains into AcrIIC1 dramatically enhances inhibition of the natural Neisseria meningitidis Cas9 target. Then, using structure-guided design, we converted AcrIIC1 into AcrX, a potent inhibitor of the type II-A CRISPR-Cas9 from Staphylococcus aureus widely applied for in vivo genome editing. Our work introduces designer Acrs as important biotechnological tools and provides an innovative strategy to safeguard the CRISPR technology.

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