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Structure and engineering of miniature Acidibacillus sulfuroxidans Cas12f1

Nature Catalysis volume 6, pages 695–709 (2023)Cite this article

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Abstract

The miniature CRISPR-Cas12f nucleases enable efficient delivery via cargo-size-limited vehicles, thereby showing promise for in vivo therapeutic applications. Acidibacillus sulfuroxidans Cas12f1 (AsCas12f1, 422 amino acids) is one of the most compact Cas12f nucleases and exhibits moderate genome-editing activity in human cells compared with Cas9 and Cas12a. Understanding the mechanisms of why such a compact nuclease is active for genome editing would facilitate its rational engineering. Here we show the cryo-electron microscopy structure of the AsCas12f1–sgRNA–dsDNA ternary complex, and reveal that AsCas12f1 functions as an asymmetric dimer for sgRNA binding and DNA targeting. The mechanisms of dimer formation, protospacer adjacent motif recognition and sgRNA accommodation are elucidated. Based on these findings, we extensively engineer this system and have produced an evolved AsCas12f1–sgRNA combination with drastically enhanced genome-editing activity in human cells. These results provide further understanding of compact CRISPR systems and expand the mini CRISPR toolbox for therapeutic applications.

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Fig. 1: Cryo-EM structure of the AsCas12f1–sgRNA–dsDNA ternary complex.
Fig. 2: Structural comparison between AsCas12f1 and Un1Cas12f1.
Fig. 3: Molecular interactions in the AsCas12f1 homodimer.
Fig. 4: PAM recognition mechanism.
Fig. 5: Architecture of the sgRNA and its engineering.
Fig. 6: Structure-directed engineering of AsCas12f1.
Fig. 7: Systematic evaluation of the engineered CRISPR-AsCas12f1 system.

Data availability

The structure of AsCas12f1 in complex with sgRNA and target DNA has been deposited in the Protein Data Bank under the accession code 7WJU, and in the EMDB under the accession code EMD-32548. All other data are available from the corresponding authors. Source data are provided with this paper.

Code availability

The sequencing data for evaluating indel efficiencies are demultiplexed by using barcodeSpliter (https://github.com/atlasbioinfo/barcodeSpliter). The indel efficiencies are evaluated by using the CRISPResso2 suite which is available at GitHub (https://github.com/pinellolab/CRISPResso2). The computational pipeline for GUIDE-seq is available at GitHub (https://github.com/aryeelab/guideseq).

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Acknowledgements

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, 2207783 and 22207074 from the National Natural Science Foundation of China; 22ZR1480100 and 22YF1428100 from the Shanghai Committee of Science and Technology, China; KF-202303 from the Open Research Fund of the National Center for Protein Sciences at Peking University in Beijing; and EKPG21-18 from the Emergency Key Program of Guangzhou Laboratory. The authors also thank the Analytical Instrumentation Center, SPST, ShanghaiTech University for technical support with the ICP-OES.

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Author notes

  1. These authors contributed equally: Zhaowei Wu, Dongliang Liu, Deng Pan.

Authors and Affiliations

  1. School of Physical Science and Technology, ShanghaiTech University, Shanghai, China

    Zhaowei Wu, Deng Pan, Jin Shi, Jiacheng Ma, Wenhan Fu, Zhipeng Wang, Fan Li, Weizhong Chen & Quanjiang Ji

  2. Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, China

    Dongliang Liu, Zijie Zheng, Yannan Qu & Huaizong Shen

  3. Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, China

    Dongliang Liu, Zijie Zheng, Yannan Qu & Huaizong Shen

  4. Department of Cell and Developmental Biology, John Innes Centre, Norwich Research Park, Norwich, UK

    Haopeng Yu

  5. Gene Editing Center, School of Life Science and Technology, ShanghaiTech University, Shanghai, China

    Xingxu Huang & Quanjiang Ji

  6. Guangzhou Laboratory, Guangzhou, China

    Xingxu Huang

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Contributions

Z. Wu and Q.J. conceived the initial study. Z. Wu, D.L., Z.Z., Y.Q. and H.S. determined the structure of the AsCas12f1 effector complex. Z. Wu, D.P., J.S., J.M., W.F., Z. Wang, F.L. and W.C. performed plasmid construction, protein purification and biochemical experiments. Z. Wu, D.P., J.S., J.M., W.F. and H.Y. performed the genome editing in human cells and NGS data analyses. Z. Wu, D.P., D.L., X.H., H.S. and Q.J analysed and discussed the experimental data. Z. Wu and Q.J. prepared the figures and wrote the paper. The paper was reviewed and approved by all coauthors.

Corresponding authors

Correspondence to Huaizong Shen or Quanjiang Ji.

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Competing interests

Q.J., Zhaowei Wu and D.P. have filed a patent application (PCT/CN2022/113357) related to this work through ShanghaiTech University. The remaining authors declare no competing interest.

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Nature Catalysis thanks Feng Gu, Ervin Welker, and Ning Jia for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Table 1 and Supplementary Figs. 1-13.

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Supplementary Data 1 and 2

Information for DNA and RNA sequences used in this study.

Supplementary Data 3

Statistical source data and unmodified gel images for Supplementary Figures.

Source data

Source Data Figs. 3–7

Statistical source data for Figs. 3c, 4d, 5e,f, 6a,c,d and 7.

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Wu, Z., Liu, D., Pan, D. et al. Structure and engineering of miniature Acidibacillus sulfuroxidans Cas12f1. Nat Catal 6, 695–709 (2023). https://doi.org/10.1038/s41929-023-00995-4

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