Abstract
CRISPR-Cas13d RNA nucleases are powerful tools for programmable RNA targeting. A light-controlled RNA nuclease could be transformative by enabling researchers to selectively knock down transcripts at desired positions in a cell or tissue or at timepoints of interest. Here, we develop a set of RfxCas13d tools that can be multimodally controlled by either light or small molecule addition. By screening an RfxCas13d library containing insertions of the AsLOV2 photoswitchable domain, we identify an OptoCas13d-off variant that induced target RNA cleavage in the dark and switched to an inactive state under blue light. We show that the same allosteric hotspot can be exploited to generate an OptoCas13d-on with an inverted light response and a ChemoCas13d that is activated by rapamycin analogs, enabling knockdown of endogenous mRNA and protein targets. Overall, our study shows that engineered allostery can produce stimulus-controlled Cas13d variants to modulate RNA with high spatial and temporal precision.
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Acknowledgments
We thank all members of the Toettcher and Myhrvold labs for helpful discussions. Molecular graphics and analyses performed with UCSF ChimeraX, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from National Institutes of Health R01-GM129325 and the Office of Cyber Infrastructure and Computational Biology, National Institute of Allergy and Infectious Diseases.
Funding
This work was supported by the Omenn-Darling Bioengineering Institute (to L.T.N.), the National Institutes of Health grants R01GM144362 and U01DK127429 (to J.E.T.), R01AI182281 (to C.M.), and T32GM007388 and T32GM148739 (to A.G.B. and K.M.G.), as well as a Princeton AI Lab Seed Grant 2025-74 (to C.M. and J.E.T.).
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J.E.T. is a scientific advisor for Prolific Machines and Nereid Therapeutics. L.Z., L.T.N., C.M., and J.E.T. have submitted a provisional patent application on allosterically controlled Cas13d variants (US Patent Application No. 63/785,347). The remaining authors declare no competing interests.
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Zhu, L., Nguyen, L.T., Bell, A.G. et al. Multimodal control of Cas13d activity through domain insertion at an allosteric hotspot. Nat Commun (2026). https://doi.org/10.1038/s41467-026-73645-5
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DOI: https://doi.org/10.1038/s41467-026-73645-5
