Abstract
Eukaryotic Fanzor proteins are compact, programmable RNA-guided nucleases with substantial potential for genome editing, although their efficiency in mammalian cells remains suboptimal. Here, we present a combinatorial engineering strategy to optimize a representative Fanzor system, MmeFz2–ωRNA. AlphaFold3-powered rational redesign produced a minimized ωRNA scaffold that is 30% smaller while maintaining up to 82.2% efficiency. Synergistic structure-guided and AI-augmented protein engineering generated two variants, enMmeFz2 and evoMmeFz2, which exhibited an average ~32-fold increase in activity across 38 genomic loci. Moreover, fusion of the non-specific DNA-binding domain HMG-D further enhanced editing performance (enMmeFz2-HMG-D and evoMmeFz2-HMG-D). Notably, evoMmeFz2-HMG-D demonstrated robust in vivo genome editing activity, enabling dystrophin restoration in humanized male Duchenne muscular dystrophy mouse models via single adeno-associated virus (AAV) delivery. This study establishes Fanzor2 as a gene editing platform for genome engineering and therapeutic applications, and underscores the power of AI-guided engineering to accelerate genome editor development while reducing experimental burden.
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Data availability
Next-generation sequencing data are available at the National Center for Biotechnology Information (NCBI) Sequence Read Archive database under the BioProject accession code PRJNA1259048. Source data are provided with this paper.
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Acknowledgements
We are grateful for the support from the Gene Editing Scientific Teaching (NWAFU-GEST), High-Performance Computing (HPC), and Life Science Research Core Service platforms (K.R. Huang, X.R. Liu, L. Chen, M. Zhou, and L.Q. Li) at Northwest A&F University (NWAFU). The authors also wish to express their gratitude to the members of HuidaGene Therapeutics Co., Ltd. for their contributions in supplying experimental materials and insightful discussions. This work is supported by the National Natural Science Foundation of China (32441080, 32301251 to Y.W. and 22207074 to Z.W.), the Biological Breeding-Major Projects (2023ZD04074 to K.X., 2023ZD04051 to Y.W., and 2022ZD04014 to X.W.), the National Key Research and Development Program of China (2023YFF1000904 to X.W.), the National Science and Technology Major Project of China (2023ZD0500500 to Z.W.), the China Agricultural Research System (CARS-39-03 to X.W.), and local grants (2024A02004-1-3, 2025NC-YBXM-109, and QCYRCXM-2023-104 to Y.W. and 2023A02011-2 to X.W.).
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Y.W., Z.W.W., K.X., and X.W. conceived the project. Y.W., Z.W.W., K.X., and X.L. designed the experiments. Y.W., S.L., P.G., and G.L. performed data analysis. Z.W.W. conducted the structural prediction analysis. S. L., P.G., Z.M.W., Y.Y., H.J., Y.F.C., Z.L., B.Z., and M.Z. performed cell transfection and FACS. Y.W. and G.L. performed animal experiments. Y.W. and Z.W.W. wrote the manuscripts. Y.W., X.W., Z.W.W., Y.L.C., K.X., and X.L. supervised the project.
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Li, S., Xu, K., Li, G. et al. Engineering the MmeFz2-ωRNA system for efficient genome editing through an integrated computational-experimental framework. Nat Commun (2026). https://doi.org/10.1038/s41467-026-68644-5
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DOI: https://doi.org/10.1038/s41467-026-68644-5
