Abstract
CRISPR–Cas12f nucleases are currently one of the smallest genome editors, exhibiting advantages for efficient delivery via cargo-size-limited adeno-associated virus delivery vehicles. Most characterized Cas12f nucleases recognize similar T-rich protospacer adjacent motifs (PAMs) for DNA targeting, substantially restricting their targeting scope. Here we report the cryogenic electron microscopy structure and engineering of a miniature Clostridium novyi Cas12f1 nuclease (CnCas12f1, 497 amino acids) with rare C-rich PAM specificity. Structural characterizations revealed detailed PAM recognition, asymmetric homodimer formation and single guide RNA (sgRNA) association mechanisms. sgRNA engineering transformed CRISPR–CnCas12f1, which initially was incapable of genome targeting in bacteria, into an effective genome editor in human cells. Our results facilitate further understanding of CRISPR–Cas12f1 working mechanism and expand the mini-CRISPR toolbox.
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Data availability
The structure of the CnCas12f1–sgRNA–dsDNA complex has been deposited in the Protein Data Bank under accession code 8HR5. Next-generation sequencing data are available at the National Center for Biotechnology Information Sequence Read Archive under accession codes PRJNA990823, PRJNA967838, PRJNA967688, PRJNA970104 and PRJNA970076. Plasmids can be accessed in Addgene under accession codes 204996 and 204999. Source data are provided with this paper.
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Acknowledgements
The authors thank the support from H. Yang at ShanghaiTech University and the Bio-Electron Microscopy facility at ShanghaiTech University for cryo-EM data collections. This work was supported by grants 2022YFC3400200 from the National Key R&D Program of China; LG-QS-202206-05 from the Lingang Laboratory; 22277078, 22077083 and 22207074 from the National Natural Science Foundation of China; 22ZR1480100 and 22YF1428100 from the Shanghai Committee of Science and Technology; and KF-202303 from the Open Research Fund of the National Center for Protein Sciences at Peking University.
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Q.J. conceived the study. M.S. performed in vitro DNA cleavage, bacterial self-targeting assays and qRT–PCR experiments. F.L., Y.G., W.L., Z.S., C.Z., N.T., J.G. and Z.W. prepared the cryo-EM sample and determined the structure. F.L. performed the PAM depletion and RNA-seq experiments. Y.W. performed the genome editing assay of HEK293T cells. M.S., F.L., Y.W., Z.W. and Q.J. analyzed and discussed the experimental data. M.S., F.L., Y.W., Z.W. and Q.J. prepared the figures and wrote the manuscript. The manuscript was reviewed and approved by all coauthors.
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Q.J., M.S. and Y.W. have filed a patent application related to this work through ShanghaiTech University (2023102864248). The remaining authors declare no competing interests.
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Nature Chemical Biology thanks Ailong Ke, Yong-Sam Kim and Hiroshi Nishimasu for their contribution to the peer review of this work.
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Supplementary Figs. 1–14 and Supplementary Tables 1–4
Supplementary Video 1
PAM-interacting residue density map
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Statistical source data and unmodified gel images for supplementary figures
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Source Data Figs. 1, 4, 5 and 6
Unmodified gel images for Figs. 1b,d, 4b, 5d,f,h and 6b
Source Data Figs. 1, 4, 5 and 6
Statistical source data for Figs. 1a, 4b, 5d,f,h and 6d–g
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Su, M., Li, F., Wang, Y. et al. Molecular basis and engineering of miniature Cas12f with C-rich PAM specificity. Nat Chem Biol 20, 180–189 (2024). https://doi.org/10.1038/s41589-023-01420-4
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DOI: https://doi.org/10.1038/s41589-023-01420-4
