Optogenetic control of Neisseria meningitidis Cas9 genome editing using an engineered, light-switchable anti-CRISPR protein
Optogenetic control of CRISPR-Cas9 systems has significantly improved our ability to perform genome perturbations in living cells with high precision in time and space. As new Cas orthologues with advantageous properties are rapidly being discovered and engineered, the need for straightforward strat...
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| Main Authors: | , , , , , , , , , |
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| Format: | Article (Journal) |
| Language: | English |
| Published: |
2021
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| In: |
Nucleic acids research
Year: 2021, Volume: 49, Issue: 5, Pages: e29-e29 |
| ISSN: | 1362-4962 |
| DOI: | 10.1093/nar/gkaa1198 |
| Online Access: | Verlag, lizenzpflichtig, Volltext: https://doi.org/10.1093/nar/gkaa1198 Verlag, lizenzpflichtig, Volltext: https://academic.oup.com/nar/article/49/5/e29/6039921 
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| Author Notes: | Mareike D Hoffmann, Jan Mathony, Julius Upmeier zu Belzen, Zander Harteveld, Sabine Aschenbrenner, Christina Stengl, Dirk Grimm, Bruno E Correia, Roland Eils and Dominik Niopek |
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| 520 | |a Optogenetic control of CRISPR-Cas9 systems has significantly improved our ability to perform genome perturbations in living cells with high precision in time and space. As new Cas orthologues with advantageous properties are rapidly being discovered and engineered, the need for straightforward strategies to control their activity via exogenous stimuli persists. The Cas9 from Neisseria meningitidis (Nme) is a particularly small and target-specific Cas9 orthologue, and thus of high interest for in vivo genome editing applications. Here, we report the first optogenetic tool to control NmeCas9 activity in mammalian cells via an engineered, light-dependent anti-CRISPR (Acr) protein. Building on our previous Acr engineering work, we created hybrids between the NmeCas9 inhibitor AcrIIC3 and the LOV2 blue light sensory domain from Avena sativa. Two AcrIIC3-LOV2 hybrids from our collection potently blocked NmeCas9 activity in the dark, while permitting robust genome editing at various endogenous loci upon blue light irradiation. Structural analysis revealed that, within these hybrids, the LOV2 domain is located in striking proximity to the Cas9 binding surface. Together, our work demonstrates optogenetic regulation of a type II-C CRISPR effector and might suggest a new route for the design of optogenetic Acrs. | ||
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