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Anti-CRISPR Proteins and Their Application to Control CRISPR Effectors in Mammalian Systems

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Mammalian Synthetic Systems

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2774))

Abstract

CRISPR-Cas effectors are powerful tools for genome and transcriptome targeting and editing. Naturally, these protein–RNA complexes are part of the microbial innate immune system, which emerged from the evolutionary arms race between microbes and phages. This coevolution has also given rise to so-called anti-CRISPR (Acr) proteins that counteract the CRISPR-Cas adaptive immunity. Acrs constitutively block cognate CRISPR-Cas effectors, e.g., by interfering with guide RNA binding, target DNA/RNA recognition, or target cleavage. In addition to their important role in microbiology and evolution, Acrs have recently gained particular attention for being useful tools and switches to regulate or fine-tune the activity of CRISPR-Cas effectors. Due to their commonly small size, high inhibition potency, and structural and mechanistic versatility, Acrs offer a wide range of potential applications for controlling CRISPR effectors in heterologous systems, including mammalian cells.

Here, we review the diverse applications of Acrs in mammalian cells and organisms and discuss the underlying engineering strategies. These applications include (i) persistent blockage of CRISPR-Cas function to create write-protected cells, (ii) reduction of CRISPR-Cas off-target editing, (iii) focusing CRISPR-Cas activity to specific cell types and tissues, (iv) spatiotemporal control of CRISPR effectors based on engineered, opto-, or chemogenetic Acrs, and (v) the use of Acrs for selective binding and detection of CRISPR-Cas effectors in complex samples. We will also highlight potential future applications of Acrs in a biomedical context and point out present challenges that need to be overcome on the way.

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Acknowledgments

We thank the research group of Dominik Niopek for feedback on this manuscript. C.M.G. is a member of the Graduate School Life Science Engineering at TU Darmstadt. D.N. acknowledges funding by the German Research Foundation (DFG) [project no. 453202693], the Schwiete Stiftung, and the Aventis Foundation.

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Authors and Affiliations

  1. Centre for Synthetic Biology, Department of Biology, Technical University Darmstadt, Darmstadt, Germany

    Carolin Maja Gebhardt

  2. Institute of Pharmacy and Molecular Biotechnology (IPMB), Faculty of Engineering Sciences, Heidelberg University, Heidelberg, Germany

    Dominik Niopek

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  1. Carolin Maja Gebhardt
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  2. Dominik Niopek
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Correspondence to Dominik Niopek .

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Editors and Affiliations

  1. Department of Chemical Engineering and Imperial College Centre for Synthetic Biology, Imperial College London, London, UK

    Francesca Ceroni

  2. Department of Chemical Engineering and Imperial College Centre for Synthetic Biology, Imperial College London, London, UK

    Karen Polizzi

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Gebhardt, C.M., Niopek, D. (2024). Anti-CRISPR Proteins and Their Application to Control CRISPR Effectors in Mammalian Systems. In: Ceroni, F., Polizzi, K. (eds) Mammalian Synthetic Systems. Methods in Molecular Biology, vol 2774. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3718-0_14

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  • DOI: https://doi.org/10.1007/978-1-0716-3718-0_14

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