Structure and evolution-guided design of minimal RNA-guided nucleases

Skopintsev, P.; Esain-Garcia, I.; DeTurk, E. C.; Yoon, P. H.; Zhou, Z.; Weiss, T.; Kamalu, M.; Chamraj, A.; Loi, K. J.; Langeberg, C. J.; Boger, R.; Nisonoff, H.; Karp, H. M.; Chen, L.; Shi, H.; Vohra, K.; Banfield, J. F.; Cate, J. H. D.; Jacobsen, S. E.; Doudna, J. A.

doi:10.64898/2025.12.08.692503

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bioRxiv · 10.64898/2025.12.08.692503

Structure and evolution-guided design of minimal RNA-guided nucleases

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Abstract

The design of RNA-guided nucleases with properties not limited by evolution can expand programmable genome editing capabilities. However, generating diverse multi-domain proteins with robust enzymatic properties remains challenging. Here we use an artificial intelligence-driven strategy that couples structure-guided inverse protein folding with evolution-informed residue constraints to generate active, divergent variants of TnpB, a minimal CRISPR-Cas12-like nuclease. High-throughput functional screening of AI-generated variants yielded editors that retained or exceeded wild-type activity in bacterial, plant and human cells. Cryo-EM-based structure determination of the most divergent active variant revealed new stabilizing contacts in the RNA/DNA interfaces across conformational states, demonstrating the design potential of this approach. Together these results establish a strategy for creating non-natural RNA-guided nucleases and conformationally active nucleic acid binders, enlarging the designable protein space. One-sentence abstractAn evolution- and structure-conditioned model enables design of active RNA-guided nucleases with new nucleic acid contacts resolved by cryo-EM.

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Skopintsev, P., Esain-Garcia, I., DeTurk, E. C., Yoon, P. H., Zhou, Z., Weiss, T., Kamalu, M., Chamraj, A., Loi, K. J., Langeberg, C. J., Boger, R., Nisonoff, H., Karp, H. M., Chen, L., Shi, H., Vohra, K., Banfield, J. F., Cate, J. H. D., Jacobsen, S. E., Doudna, J. A.. 2025-12-08. Structure and evolution-guided design of minimal RNA-guided nucleases. https://doi.org/10.64898/2025.12.08.692503

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