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Cas1 mediates the interference stage in a phage-encoded CRISPR–Cas system

Nature Chemical Biology (2024)Cite this article

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Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR)–Cas systems are prokaryotic adaptive immune systems against invading phages and other mobile genetic elements. Notably, some phages, including the Vibrio cholerae-infecting ICP1 (International Center for Diarrheal Disease Research, Bangladesh cholera phage 1), harbor CRISPR–Cas systems to counteract host defenses. Nevertheless, ICP1 Cas8f lacks the helical bundle domain essential for recruitment of helicase-nuclease Cas2/3 during target DNA cleavage and how this system accomplishes the interference stage remains unknown. Here, we found that Cas1, a highly conserved component known to exclusively work in the adaptation stage, also mediates the interference stage through connecting Cas2/3 to the DNA-bound CRISPR-associated complex for antiviral defense (Cascade; CRISPR system yersinia, Csy) of the ICP1 CRISPR–Cas system. A series of structures of Csy, Csy–dsDNA (double-stranded DNA), Cas1–Cas2/3 and Csy–dsDNA–Cas1–Cas2/3 complexes reveal the whole process of Cas1-mediated target DNA cleavage by the ICP1 CRISPR–Cas system. Together, these data support an unprecedented model in which Cas1 mediates the interference stage in a phage-encoded CRISPR–Cas system and the study also sheds light on a unique model of primed adaptation.

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Fig. 1: Cas1 mediates target DNA cleavage by connecting the Csy–dsDNA complex to Cas2/3.
Fig. 2: Structure of the ICP1 Csy complex and Csy–dsDNA complex.
Fig. 3: Cryo-EM structure of the ICP1 Csy–dsDNA–Cas1–Cas2/3 complex.
Fig. 4: Structural plasticity of the ICP1 Cas1–Cas2/3 complex.
Fig. 5: The ICP1 Cas1 dimer connects Cas2/3 with the Csy–dsDNA complex.
Fig. 6: ICP1 Cas2/3 binds to Cas5f and captures the extended NTS.

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Data availability

The atomic coordinates for the crystal structures of the Csy complex (PDB 8K0K), Cas5f–Cas8f (PDB 8K0H) and Cas7f (PDB 8K0J) and cryo-EM structures of the ICP1 Csy–dsDNA complex (partial duplex) (PDB 8K27), ICP1 Csy–dsDNA complex (form 1) (PDB 8K28), ICP1 Csy–dsDNA complex (form 2) (PDB 8K29), ICP1 Cas1–Cas2/3–dsDNA complex (PDB 8K25), ICP1 Csy–dsDNA–Cas1–Cas2/3 complex (C1, fully assembled form) (PDB 8K23), ICP1 Csy–dsDNA–Cas1–Cas2/3 complex (C2, fully assembled form) (PDB 8K24), ICP1 Csy–dsDNA–Cas1–Cas2/3 complex (half form) (PDB 8K22), Cas1–Cas2–dsDNA subregion in ICP1 Csy–DNA–Cas1–Cas2/3 complex (PDB 8K21) and ICP1 Cas1–Cas2/3 complex (PDB 8K26) were deposited to the PDB (www.rcsb.org). The cryo-EM density maps reported in this study for the ICP1 Csy–dsDNA complex (partial duplex) (EMD-36830), ICP1 Csy–dsDNA complex (form 1) (EMD-36831), ICP1 Csy–dsDNA complex (form 2) (EMD-36835), ICP1 Cas1–Cas2/3–dsDNA complex (EMD-36828), ICP1 Csy–dsDNA–Cas1–Cas2/3 complex (C1, fully assembled form) (EMD-36826), ICP1 Csy–dsDNA–Cas1–Cas2/3 complex (C2, fully assembled form) (EMD-36827), ICP1 Csy–dsDNA–Cas1–Cas2/3 complex (half form) (EMD-36825), Cas1–Cas2–dsDNA subregion in ICP1 Csy–DNA–Cas1–Cas2/3 complex (EMD-36824), Csy1 subregion in ICP1 Csy–DNA–Cas1–Cas2/3 complex (EMD-36832), Cas3 subregion in ICP1 Csy–DNA–Cas1–Cas2/3 complex (EMD-36833), Csy4 subregion in ICP1 Csy–DNA–Cas1–Cas2/3 complex (EMD-36834) and ICP1 Cas1–Cas2/3 complex (EMD-36829) were deposited to the EM Data Bank. Source data are provided with this paper.

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Acknowledgements

We thank J. Lei and F. Yang (Tsinghua University) and P. Wang and X. Ma (Southern University of Science and Technology) for EM data collection. We thank the Tsinghua University Branch of the China National Center for Protein Sciences (Beijing) and Southern University of Science and Technology for providing the cryo-EM facility support. We thank the staff at beamlines BL17U1 and BL19U1 of the Shanghai Synchrotron Radiation Facility for their assistance with data collection. We thank the Tsinghua University Branch of China National Center for Protein Sciences Beijing and S. Fan for providing facility support for X-ray diffraction of the crystal samples. We thank W. Yao (State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences) for assistance in the MST experiment. This work was supported by the National Key Research and Development Program of China (2022YFC2104800), the National Natural Science Foundation of China (32371329, 32171274 and 32030056), the Tsinghua-Foshan Innovation Special Fund (TFISF-2022THFS6122), the King Abdullah University of Science and Technology Office of Sponsored Research (OSR-2020-CRG9-4352), the Beijing Nova program (20220484160), the Fundamental Research Funds for the Central Universities (QNTD2023-01), the China Postdoctoral Science Foundation (CPSF; 2023M740203 and 2023TQ0019) and the Postdoctoral Fellowship Program of CPSF (GZB20230051 and GZC20230208).

Author information

Author notes

  1. These authors contributed equally: Laixing Zhang, Hao Wang, Jianwei Zeng, Xueli Cao, Zhengyu Gao.

Authors and Affiliations

  1. Ministry of Education Key Laboratory of Protein Science, Beijing Advanced Innovation Center for Structural Biology, Beijing Frontier Research Center for Biological Structure, Tsinghua-Peking Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing, China

    Laixing Zhang, Jianwei Zeng, Jiawei Wang & Maojun Yang

  2. Beijing Advanced Innovation Center for Soft Matter Science and Engineering, Beijing Key Laboratory of Bioprocess, State Key Laboratory of Chemical Resource Engineering, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, China

    Hao Wang, Xueli Cao, Zhengyu Gao, Zihe Liu, Feixue Li, Yi Zhang & Yue Feng

  3. SUSTech Cryo-EM Facility Center, Southern University of Science and Technology, Shenzhen, China

    Maojun Yang

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Contributions

Y.F. conceptualized and supervised the project. H.W., X.C., Z.G., Z.L. and F.L. purified the proteins, prepared the protein–nucleic acid complexes and performed the activity analysis and binding assays supervised by Y.F. and Y.Z. L.Z. prepared the cryo-EM samples, collected the cryo-EM data and solved the cryo-EM structures supervised by M.Y. H.W. collected the diffraction data of crystals. J.Z. solved the crystal structures supervised by J.W. Y.F. helped solve the crystal structures. Y.F. analyzed the data and wrote the paper with assistance from all the authors.

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Correspondence to Yi Zhang, Maojun Yang or Yue Feng.

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Nature Chemical Biology thanks Ning Jia and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 ICP1 Cas1 forms a complex with Csy-dsDNA and Cas2/3.

a, Sequence alignment of full-length ICP1 Cas8f and PaeCas8f (the region of 1-282 aa). Identical residues are colored in dark blue. b, Protein binding assays. 1 µM Pae Csy was incubated with 4 µM PaeCas2/3 only, or first incubated with 2 µM PaeCas1 followed by adding 4 µM Cas2/3. The mixture was separated by native PAGE and visualized by Coomassie blue R250 staining. c, In vitro DNA cleavage of the Pae CRISPR-Cas system. 0.04 µM dsDNA was preincubated with 0.4 µM Csy complex. Next, Cas1-Cas2/3 (0.04/0.08/0.16 µM) or Cas2/3 (0.08/0.16/0.32 µM) with 1 mM ATP and 5 mM MgCl2, 5 mM CaCl2 and 75 µM NiSO4 were added into the reaction system. The reaction was terminated at 60 min. The products were separated by Urea-PAGE and visualized by fluorescence imaging. d, Gel filtration chromatography of ICP1 Csy-dsDNA-Cas1-Cas2/3 complex. e, Fractions (red box in d) were analyzed by SDS-PAGE and native PAGE in the right panels. f, Purified ICP1 Cas1-Cas2/3 complex and mutants were analyzed by SDS-PAGE. g, Sequence alignment of Cas1. The first 7 Cas1s are associated with short Cas8s, the last one is associated with long Cas8. h, 1.2 µM dsDNA was pre-incubated with 8 µM Csy complex. Next, Cas2/3 (10 µM) with 3 mM ATP and 4 mM MnCl2 and NiSO4 were added into the reaction system. The reaction was terminated at 60 min. The products were separated by Urea-PAGE and visualized by fluorescence imaging.

Source data

Extended Data Fig. 2 Plaque assays of the ICP1 CRISPR-Cas system. Related to Figure 1h and 5d.

The experiment has been repeated independently for 3 times and a representative result is shown. Cas2/3 M in this figure represents Cas2/3 D112A/D280A.

Source data

Extended Data Fig. 3 Cryo-EM micrograph, 2D class averages and data processing of the ICP1 Csy-dsDNA-Cas1-Cas2/3 complex.

a, Representative micrograph of the sample of ICP1 Csy-dsDNA-Cas1-Cas2/3 complex. The scale bar represents 20 nm. b, Representative 2D class averages of the sample of ICP1 Csy-dsDNA-Cas1-Cas2/3 complexes. c, Cryo-EM image-processing flow-chart of ICP1 Csy-dsDNA-Cas1-Cas2/3 complex. d, Image-processing procedure of ICP1 Cas1-Cas2/3 complex. The scale bar represents 10 nm.

Extended Data Fig. 4 Local resolution estimation of the cryo-EM structures of the ICP1 Csy complexes, Cas1-Cas2/3 complexes and Csy-dsDNA-Cas1-Cas2/3 complexes.

a, Overview of data collection and image-processing procedure of ICP1 Csy-dsDNA complex (partial duplex) (see Methods). The scale bar represents 10 nm. b, Image processing procedure, local resolution maps, angular distribution, 3DFSC analyses and model-to-map FSC curves (FSC = 0.5) were shown for ICP1 Csy-dsDNA complexes (form 1 and form2) and Csy-dsDNA Cas1-Cas2/3 complexes (fully assembled form and half form). c, Gold-standard FSC curves (FSC = 0.143) of the final 3D reconstructions of ICP1 Csy complexes, Cas1-Cas2/3 complexes and Csy-dsDNA-Cas1-Cas2/3 complexes. d, Local resolution maps, angular distribution and 3DFSC analyses were shown for the ICP1 Cas1-Cas2/3 and Cas1-Cas2/3-dsDNA complex. e, Local resolution maps, angular distribution and 3DFSC analyses were shown for the subregion maps of Csy1, Cas3, Csy4 and Cas1-Cas2/3 for ICP1 Csy-dsDNA-Cas1-Cas2/3 complex.

Extended Data Fig. 5 Structural characteristics of ICP1 Csy and comparison with Pae Csy.

a, 2Fo-Fc electron density of the Cas6f subunit and crRNA 3’ hairpin in the crystal structure of Csy complex contoured at 1 σ. b, The crRNA in ICP1 Csy exhibits distortions (kinks) along the Cas7f backbone at regular 6-nucleotide intervals. c–e, Structural alignment of ICP1 Cas5f and PaeCas5f (c), ICP1 Cas7f and PaeCas7f (d) and ICP1 Cas6f and PaeCas6f (e). ICP1 Cas7f has shorter ‘thumbs’ and ‘webs’ regions than PaeCas7f (d).

Extended Data Fig. 6 Structural characteristics of ICP1 Csy-dsDNA and comparisons with Pae Csy-dsDNA.

a, Structural alignment of ICP1 Csy-partial dsDNA and Csy-full dsDNA (full R-loop form), they are colored in slate and tv_orange, respectively. b, Details of target DNA PAM recognition in the Cas8f and Cas5f region. c, crRNA:target DNA heteroduplex and duplex DNA are shown in cartoon model. There is one kinked-off nucleotide at every 6th position in the crRNA:target DNA heteroduplex. d, NTS is attached to the positively charged groove of the Cas8f subunit. e, Target DNA strand is locked by Cas8f-HB, the ‘thumb’ and ‘web’ regions of Cas7.2 f and Cas7.3 f in the Pae Csy-dsDNA complex (PDB code: 6NE0). Two views are shown. f, K58 and K60 of Cas7f, two critical residues for dsDNA binding in PaeCas7f, is lacking in ICP1 Cas7f.

Extended Data Fig. 7 Cas1-Cas2/3 binds the Csy-dsDNA form but not the apo Csy.

a, Superimposition of the apo Csy onto the Csy within the Csy-dsDNA-Cas1-Cas2/3 complex at the Cas6f subunit (in the left side of the complex). Two views are shown. Close view of the regions in boxes in the left side are shown in the right side. b, Structural alignment between the Csy-dsDNA complex (colored slate) and that within the Csy-dsDNA-Cas1-Cas2/3 complex. Two views are shown.

Extended Data Fig. 8 Structural comparisons of ICP1 Cas1 and Cas2/3 with their Pae homologs.

a, Purified ICP1 Cas2/3 and Cas1-Cas2/3 complex were analyzed by SDS-PAGE. b, Structural comparison between apo ICP1 Cas1-Cas2 and that in Cas1-Cas2-dsDNA complex. The Cas2 dimer is superimposed. Vector length correlates with the domain motion scale. The black arrows indicate domain movements within Cas1-Cas2 complex upon dsDNA binding. c, Structural comparison of ICP1 Cas1.1 in Cas1-Cas2-dsDNA complex and that in Csy-dsDNA-Cas1-Cas2/3 complex. d, Static light scattering (SLS) study of ICP1 Cas1. The calculated molecular weight of the main peak is shown. e, Structural comparison of ICP1 Cas1 dimer in Csy-dsDNA-Cas1-Cas2/3 complex and apo Cas1 dimer (PDB code: 4W8K, gray). f, Structural comparison of ICP1 Cas1.2 in Csy-dsDNA-Cas1-Cas2/3 complex and PaeCas1.2 (PDB code: 3GOD, gray). g–j, Structural comparison of the Cas2 domains (g), HD domains (h), RecA1 domains (i), and the region of RecA2/linker/CTD (j) of ICP1 Cas2/3 and PaeCas2/3 (PDB code: 5B7I, gray). The active site residues of the HD domain of ICP1Cas2/3, D112 and D280 correspond to D124 and D315 of PaeCas2/3, respectively. The active site of the RecA1 helicase, D510 of ICP1Cas2/3 also stands at the same position as D576 of PaeCas2/3. k, Structural comparison of ICP1 Cas1-2/3 (left) and PaeCas1-2/3 (right, PDB code: 8FLJ). Cas2 domain of ICP1 and Pae are aligned. The distance between the ICP1 Cas1.1/1.2 and PaeCas1.1/1.2 mass centers is 1.74 Å, and between the ICP1 Cas1.1’/1.2’ and PaeCas1.1’/1.2’ is 3.78 Å. The distance between ICP1 Cas2/3 and PaeCas2/3 mass centers is 35.75 Å, and between the ICP1 Cas2/3’ and PaeCas2/3’ is 28.84 Å.

Source data

Extended Data Fig. 9 Comparison of structure of ICP1 dsDNA-Csy-Cas1-2/3 and the model of Pae dsDNA-Csy-Cas1-2/3.

Superimposition of ICP1 Csy complex and Pae Csy complex (PDB code: 6NE0). PaeCas1-2/3 (PDB code: 8FLJ) is docked to the PaeCas8f HB domain.

Extended Data Fig. 10 Structural details of the Csy-dsDNA-Cas1-Cas2/3 complex.

a, Superimposition of the apo Csy onto the Csy within the Csy-dsDNA-Cas1-Cas2/3 complex at the Cas6f subunit. The apo Csy structure is colored in light blue. b, The densities corresponding to Cas6f and crRNA 3’ hairpin in the Csy-dsDNA-Cas1-Cas2/3 complex are shown in mesh. Cas6f and crRNA are shown in cartoon and representative residues are shown as sticks. c, Each ICP1 Cas1.1 interacts with both two Cas2/3 protomers in the Csy-dsDNA-Cas1-Cas2/3 complex. d, Detailed interactions between Cas1.1 and Cas2/3’. Hydrogen bond and electrostatic interactions are showed as red dashed lines.

Supplementary information

Supplementary Information

Supplementary Tables 1–6: data collection and refinement statistics.

Reporting Summary

Supplementary Video 1

The apo ICP1 Csy and Csy–dsDNA structure with 32-nt modeled protospacer region.

Supplementary Video 2

The architecture of the fully assembled Csy–dsDNA–Cas1–Cas2/3 complex.

Source data

Source Data Fig. 1

Unprocessed gels.

Source Data Fig. 2

Unprocessed gels.

Source Data Fig. 5

Unprocessed gels.

Source Data Extended Data Fig. 1

Unprocessed gels.

Source Data Extended Data Fig. 2

Unprocessed photos.

Source Data Extended Data Fig. 8

Unprocessed gels.

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Zhang, L., Wang, H., Zeng, J. et al. Cas1 mediates the interference stage in a phage-encoded CRISPR–Cas system. Nat Chem Biol (2024). https://doi.org/10.1038/s41589-024-01659-5

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