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The compact Casπ (Cas12l) ‘bracelet’ provides a unique structural platform for DNA manipulation

Cell Research volume 33, pages 229–244 (2023)Cite this article

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Abstract

CRISPR-Cas modules serve as the adaptive nucleic acid immune systems for prokaryotes, and provide versatile tools for nucleic acid manipulation in various organisms. Here, we discovered a new miniature type V system, CRISPR-Casπ (Cas12l) (~860 aa), from the environmental metagenome. Complexed with a large guide RNA (~170 nt) comprising the tracrRNA and crRNA, Casπ (Cas12l) recognizes a unique 5′ C-rich PAM for DNA cleavage under a broad range of biochemical conditions, and generates gene editing in mammalian cells. Cryo-EM study reveals a ‘bracelet’ architecture of Casπ effector encircling the DNA target at 3.4 Å resolution, substantially different from the canonical ‘two-lobe’ architectures of Cas12 and Cas9 nucleases. The large guide RNA serves as a ‘two-arm’ scaffold for effector assembly. Our study expands the knowledge of DNA targeting mechanisms by CRISPR effectors, and offers an efficient but compact platform for DNA manipulation.

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Fig. 1: Identification of CRISPR-Casπ.
Fig. 2: Casπ effector cleaves dsDNA with 5′ C-rich PAM.
Fig. 3: Biochemical screening of Casπ nuclease activity.
Fig. 4: Specificity of DNA cleavage by Casπ.
Fig. 5: Casπ facilitates DNA manipulation in bacterial and human cells.
Fig. 6: The structure of Casπ nuclease.
Fig. 7: The structure of ‘bracelet’ architecture of Casπ.
Fig. 8: The structure of Casπ sgRNA.
Fig. 9: The hypothetical co-evolution trend.

Data availability

The electron density maps have been deposited to the Electron Microscopy Data Bank (EMDB) under the accession number of EMD-33983 which are publicly available as of the date of publication. The atomic coordinates and structure factors have been deposited to the Protein Data Bank (PDB) under the accession number of 7YOJ which are publicly available as of the date of publication. The raw cryo-EM micrographs and movies used in this study will be shared by corresponding author upon request. The raw sequencing result of metagenome is uploaded to NCBI database with the accession ID of PRJNA857874. Any additional information required to re-analyze the data reported in this paper is available from the corresponding author upon request.

Material availability

Plasmids generated in this study will be deposited to Addgene or are available upon request. Requests for materials should be addressed to the lead contact J.J.G.L. (junjiegogoliu@tsinghua.edu.cn).

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Acknowledgements

EM data were collected at the Tsinghua Cryo-EM facility and Shuimu Bioscience. The data were analyzed using the Bio-Computation platform at the Tsinghua University Branch of the Chinese National Center for Protein Sciences (Beijing). We thank the supports from the Tsinghua University Technology Center for Protein Research, Genome Sequencing and Analysis. We thank J.L. Lei, X.M. Li, and X.D. Li for expert electron microscopy assistance. We thank T. Yang, Y.K. Wang, A.B. Jia for computational support. We thank D. Chia, Y. Lin, and N. Liu for their kind advice on the manuscript. The work was supported by the National Key R&D Program of China (2022YFF1002801 to J.J.G.L.), the Ministry of Agriculture and Rural Affairs of China (J.J.G.L.), the National Natural Science Foundation of China (32150018 to J.J.G.L and 32101195 to S.Z.), and start-up funds from Tsinghua University, Beijing (J.J.G.L.).

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Author notes

  1. These authors contributed equally: Ao Sun, Cheng-Ping Li, Zhihang Chen, Shouyue Zhang.

Authors and Affiliations

  1. Beijing Advanced Innovation Center for Structural Biology & Frontier Research Center for Biological Structure, School of Life Sciences, Tsinghua University, Beijing, China

    Ao Sun, Cheng-Ping Li, Zhihang Chen, Shouyue Zhang, Dan-Yuan Li, Yun Yang, Long-Qi Li, Yuqian Zhao, Jia Wang & Jun-Jie Gogo Liu

  2. Tsinghua-Peking Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing, China

    Ao Sun, Cheng-Ping Li, Zhihang Chen, Shouyue Zhang, Dan-Yuan Li, Yun Yang, Long-Qi Li, Yuqian Zhao, Kaichen Wang, Zhaofu Li, Jia Wang & Jun-Jie Gogo Liu

  3. Department of Environmental Engineering, Peking University, Beijing, China

    Jinxia Liu & Sitong Liu

  4. Key Laboratory of Water and Sediment Sciences, Ministry of Education of China, Beijing, China

    Jinxia Liu & Sitong Liu

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Contributions

J.J.G.L. supervised the project. J.J.G.L., J.W., A.S., C.P.L., Z.C., S.Z., and S.L. designed the experiments. C.P.L., S.Z., S.L., and J.L. collected and analyzed the environmental metagenome. C.P.L. and S.Z. built the bioinformatics pipeline and discovered the new system. A.S. purified the Casπ proteins and performed the biochemical assays and analyses. J.W. and C.P.L. did the structural analysis and built the atomic model. Z.C., A.S., D.Y.L., Y.Y., L.Q.L., Y.Z., K.W., and Z.L. did the gene editing experiments in bacterial and mammalian cells. J.J.G.L., J.W., A.S., C.P.L., and Z.C. wrote the manuscript with help from all authors.

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Correspondence to Jia Wang or Jun-Jie Gogo Liu.

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Sun, A., Li, CP., Chen, Z. et al. The compact Casπ (Cas12l) ‘bracelet’ provides a unique structural platform for DNA manipulation. Cell Res 33, 229–244 (2023). https://doi.org/10.1038/s41422-022-00771-2

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