Abstract
The compact CRISPR-Cas12f system is promising for AAV-delivered gene therapy, but its application has been constrained by restrictive PAM recognition (e.g., TTTR) and suboptimal editing efficiency. Through bacterial library screening and mammalian cell validation, we engineer evoCas12f, an optimized variant incorporating five key mutations, that dramatically expands PAM recognition to NTNR/NYTR. This advancement reduces median distance between two neighbouring PAM sites to 2 nucleotides in the human genome. It also demonstrates 1.4-fold enhanced activity at TTTR sites compared to wild-type Un1Cas12f1, achieving up to 91% editing efficiency. Remarkably, evoCas12f enables efficient generation of homozygous mutations in F0 generation mice, even at non-canonical PAM sites. We further adapt this system for robust transcriptional activation and precise base editing with a well-defined editing window. As a compact yet highly efficient platform, evoCas12f represents a significant advance in CRISPR technology, enabling multiplexed editing for high-resolution targeting applications and expanding possibilities for therapeutic genome engineering.
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Data availability
The NGS data generated in this study have been deposited in the NCBI Sequence Read Archive (SRA) database under the accession codes: PRJNA1310035, PRJNA1310152, PRJNA1310189, PRJNA1310199, PRJNA1310212, PRJNA1310251, PRJNA1372820, PRJNA1372987.ClinVar database (ClinVar (nih.gov)) was used to identify pathogenic SNVs that can be correct by base editing. The published structure of Un1Cas12f1 (PDB ID: 7C7L) can be accessed at the RCSB Protein Data Bank (PDB) 7C7L: Cryo-EM structure of the Cas12f1-sgRNA-target DNA complex. There are no restrictions on data availability. Source data are provided with this paper.
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Acknowledgements
We thank Y. Zhang from the Flow Cytometry Core Facility of School of Life Sciences at ECNU and support from the ECNU Public Platform for innovation (011). This work was partially supported by grants from the National Key R&D Program of China (2023YFE0209200 and 2023YFC3403400 to D.L., 2022YFC3400200 to Y.G.), National Natural Science Foundation of China (32025023, 32230064 and 32311530111 to D.L., U24A20677 to L.W.), the Agriculture Science and Technology Major Project, Innovation Program of Shanghai Municipal Education Commission (2025GDZKZD03 to D.L.), Shanghai Municipal Commission for Science and Technology (24J22800400 to D.L.).D.L. is a Shanghai Academy of Natural Sciences Exploration Scholar.
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Y.H., L.W., L.Y., and D.L. conceived the rational engineering of Un1Cas12f1. Y.H., L.W., and D.C. performed the experiments of Un1Cas12f1 mutation library construction. Y.H., L.W., and J.M. performed the experiments of bacterial positive selection assay. Y.H., J.M., B.Y. performed the experiments of plasmid construction, cell culture, cell transfection, cell sorting, and genomic DNA preparation. Y. H. and J. M. performed the experiments of PCR, reverse transcription, qPCR and prepared the HTS libraries. L.W., X.W., and Dan Zhang performed computational analysis. Y.H., C.D., and S.Y. performed the experiments of lentiviral vector production and the creation of stable cell lines. Y.H., J.M., Dexin Zhang and M.L. performed the animal experiments. G.S. performed the structural analysis. L.W., Y.H., L.Y., D.L., Y.G., B.D., Y.W., H.L., and Z.Z. designed the experiments and wrote the manuscript with the input from all the authors. D.L., L.Y., and L.W. supervised the study.
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D.L., L.W, Y.H., J.M, L.Y, and Dan Zhang have submitted patent applications (application number CN 202510015341.4, under review) based on the results reported in this study. This patent mainly relates to evoCas12f in this paper. The remaining authors declare no competing interests.
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Huo, Y., Mei, J., Zhang, D. et al. Engineered Un1Cas12f1 for multiplex genome editing with enhanced activity and targeting scope. Nat Commun (2026). https://doi.org/10.1038/s41467-026-69678-5
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DOI: https://doi.org/10.1038/s41467-026-69678-5
