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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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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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.
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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Su, M., Li, F., Wang, Y. et al. Molecular basis and engineering of miniature Cas12f with C-rich PAM specificity. Nat Chem Biol (2023). https://doi.org/10.1038/s41589-023-01420-4