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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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.
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. (email@example.com).
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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.).
Tsinghua University has filed a patent that includes work described in this paper.
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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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