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Computational design of anti-CRISPR proteins with improved inhibition potency


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.

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Fig. 1: Improving the efficacy of Acr proteins by protein engineering.
Fig. 2: Domain insertion into AcrIIC1 yields a highly potent NmeCas9 inhibitor.
Fig. 3: Enhanced potency of the AcrIIC1-mCherry chimeric inhibitor arises from multiple factors.
Fig. 4: Structure-guided design of AcrIIC1X, an Acr protein targeting SauCas9.
Fig. 5: Characterization of AcrIIC1X, a designer SauCas9 inhibitor.

Data availability

Vectors encoding AcrIIC1X, AcrIIC1X* as well as the AcrIIC1-mCherry chimera will be made available via Addgene (plasmids nos. 128112–128114). Annotated vector sequences (GenBank files) are provided in the Supplementary Data 1. Additional data can be obtained from the corresponding authors on reasonable request.

Code availability

Code and data for the computational domain assembly and the design of the improved AcrIIC1 point mutants will be made available on GitHub (


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We thank the Synthetic Biology (IPMB, Heidelberg University), Virus-Host Interactions (Heidelberg University Clinics) and the Protein Design and Immunoengineering groups (EPFL, Lausanne) for helpful discussions; K. Börner (Heidelberg University Clinics, Heidelberg, Germany) and K. Rippe (DKFZ, Heidelberg, Germany) for providing cell lines; and K. Niopek for critical reading of the manuscript. We thank the EPFL’s Scientific IT and Application Support Center for their support on the computational infrastructure. We thank the Protein Production and Structure Core facility at EPFL for their support on the protein biophysical characterization experiments. This study was funded by the Helmholtz association, the German Research Foundation (DFG) and the Federal Ministry of Education and Research (BMBF) (R.E.). D.G. is grateful for funding from the German Center for Infection Research (DZIF, grant no. TTU-HIV 04.803 and grant no. TTU-HIV 04.815) and the Cystic Fibrosis Foundation Therapeutics (CFFT, grant no. GRIMM15XX0). C. Schmelas, D.G. and R.E. acknowledge funding from the Transregional Collaborative Research Center TRR179 (DFG, Projektnummer 272983813). C. Schmelas and D.G. acknowledge additional funding by the Cluster of Excellence CellNetworks (DFG, grant no. EXC81). B.E.C. is a grantee from the European Research Council (starting grant no. 716058), the Swiss National Science Foundation and the Biltema Foundation. Part of the computational simulations were performed at the CSCS Swiss National Supercomputing Centre through a grant obtained by B.E.C. Z.H. is supported by a grant from the Swiss National Science Foundation. Z.H. and S.R. are supported by a grant from the National Center of Competence in Research in Chemical Biology. Y.W. is supported by the Natural Science Foundation of China (grant nos. 31930065, 31725008, 31630015, 31571335 and 31700662).

Author information




D.N. conceived the initial idea and refined it together with J.U.z.B. and B.E.C.. J.M., C. Schmelas, C. Stengl, S.A. and D.N. designed and performed experiments. S.A. and M.D.H. performed AAV production. S.R. and S.G. purified the Acrs and performed protein-biochemical characterization. Z.H., J.U.z.B., A.S. and B.E.C. performed in silico structural analysis and modeling. W.S. and Y.W. designed and performed in vitro DNA binding and cleavage assays. D.G. provided expertise on AAVs and SauCas9. R.E. supported the computational and experimental work. B.E.C. and D.N. jointly directed the work. B.E.C. and D.N. wrote the manuscript with support from all authors.

Corresponding authors

Correspondence to Bruno E. Correia or Dominik Niopek.

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The authors declare no competing interests.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–18 and Tables 1–3.

Reporting Summary

Supplementary Data 1

Annotated vector sequences (GenBank files).

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Mathony, J., Harteveld, Z., Schmelas, C. et al. Computational design of anti-CRISPR proteins with improved inhibition potency. Nat Chem Biol 16, 725–730 (2020).

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