A Versatile Anti-CRISPR Platform for Opto- and Chemogenetic Control of CRISPR-Cas9 and Cas12 across a Wide Range of Orthologs

Brenker, L.; Aschenbrenner, S.; Bubeck, F.; Staykov, K.; Gebhardt, C.; Wolf, B.; Jendrusch, M.; Kroell, A.-S.; Mathony, J.; Niopek, D.

doi:10.1101/2024.11.25.625186

bioRxiv · 10.1101/2024.11.25.625186

A Versatile Anti-CRISPR Platform for Opto- and Chemogenetic Control of CRISPR-Cas9 and Cas12 across a Wide Range of Orthologs

Brenker, L.·Aschenbrenner, S.·Bubeck, F.·Staykov, K.·Gebhardt, C.·Wolf, B.·Jendrusch, M.·Kroell, A.-S.·Mathony, J.·Niopek, D.

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Abstract

CRISPR-Cas technologies have revolutionized life sciences by enabling programmable genome editing across diverse organisms. Achieving dynamic and precise control over CRISPR-Cas activity with exogenous triggers, such as light or chemical ligands, remains an important need. Existing tools for CRISPR-Cas control are often limited to specific Cas orthologs or selected applications, restricting their versatility. Anti-CRISPR (Acr) proteins, natural inhibitors of CRISPR-Cas systems, provide a flexible regulatory layer but are constitutively active in their native forms. In this study, we built on our previously reported concept for optogenetic CRISPR-Cas control with engineered, light-switchable anti-CRISPR proteins and expanded it from ortholog-specific Acrs towards AcrIIA5 and AcrVA1, broad-spectrum inhibitors of CRISPR-Cas9 and -Cas12a, respectively. We then conceived and implemented a novel, chemogenetic anti-CRISPR platform based on engineered, circularly permuted ligand receptor domains of human origin, that together respond to six different, clinically-relevant drugs. The resulting toolbox achieves both optogenetic and chemogenetic control of genome editing in human cells with a wide range of CRISPR-Cas effectors, including type II-A and -C CRISPR-Cas9s, and -Cas12a. In sum, this work establishes a versatile platform for multidimensional control of CRISPR-Cas systems, with immediate applications in basic research and biotechnology and potential for therapeutic use in the future.

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Brenker, L., Aschenbrenner, S., Bubeck, F., Staykov, K., Gebhardt, C., Wolf, B., Jendrusch, M., Kroell, A.-S., Mathony, J., Niopek, D.. 2024-11-25. A Versatile Anti-CRISPR Platform for Opto- and Chemogenetic Control of CRISPR-Cas9 and Cas12 across a Wide Range of Orthologs. https://doi.org/10.1101/2024.11.25.625186

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