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AcrIF5 specifically targets DNA-bound CRISPR-Cas surveillance complex for inhibition


CRISPR-Cas systems are prokaryotic antiviral systems, and phages use anti-CRISPR proteins (Acrs) to inactivate these systems. Here we present structural and functional analyses of AcrIF5, exploring its unique anti-CRISPR mechanism. AcrIF5 shows binding specificity only for the target DNA-bound form of the crRNA-guided surveillance (Csy) complex, but not the apo Csy complex from the type I-F CRISPR-Cas system. We solved the structure of the Csy–dsDNA–AcrIF5 complex, revealing that the conformational changes of the Csy complex caused by dsDNA binding dictate the binding specificity for the Csy–dsDNA complex by AcrIF5. Mechanistically, five AcrIF5 molecules bind one Csy–dsDNA complex, which destabilizes the helical bundle domain of Cas8f, thus preventing subsequent Cas2/3 recruitment. AcrIF5 exists in symbiosis with AcrIF3, which blocks Cas2/3 recruitment. This attack on the recruitment event stands in contrast to the conventional mechanisms of blocking binding of target DNA. Overall, our study reveals an unprecedented mechanism of CRISPR-Cas inhibition by AcrIF5.

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Fig. 1: AcrIF5 interacts with the Csy–DNA complex and inhibits the CRISPR system in vitro.
Fig. 2: Overall cryo-EM structure of Csy–DNA–AcrIF5 complex.
Fig. 3: Interaction mode between AcrIF5 and the Csy–dsDNA complex.
Fig. 4: Interactions between AcrIF5 and the Csy–dsDNA complex are essential for the inhibition capacity of AcrIF5.
Fig. 5: AcrIF5 binding replaces the HB of Cas8f.
Fig. 6: AcrIF5 enhances the prevention of Cas2/3 recruitment by AcrIF3.

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Data availability

The accession number for the coordinate and structure factor of AcrIF5 is PDB 7F45. The cryo-EM reconstruction of Csy–dsDNA–AcrIF5 has been deposited in the Electron Microscopy Data Bank under accession no. EMD-31265. Coordinates for the atomic model of Csy–dsDNA–AcrIF5 have been deposited in the Protein Data Bank under accession no. 7EQG. Structures of aPelota from A. pernix K1 (PDB 6JI2), Cas12i2 (PDB 6LTP), Tex protein from P. aeruginosa (PDB 3BZC) and Csy–dsDNA (PDB 6NE0) are referenced in the paper. Source data are provided with this paper.


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We thank P. Wang at Southern University of Science and Technology for data collection. We acknowledge support with cryo-EM facilities from the Southern University of Science and Technology. We acknowledge the Protein Preparation and Characterization Core Facility of Tsinghua University Branch of China National Center for Protein Sciences Beijing and D. Li and Y. Li for providing the facility support. This work was supported by the National Natural Science Foundation of China (grants nos. 32171274 and 31822012), the National Key Research and Development Program of China (grant no. 2017YFA0506500), Beijing Nova program, the Fundamental Research Funds for the Central Universities (grant no. XK1802-8) to Y.F., the National Natural Science Foundation of China (grant no. 32000901), the National Key Research and Development Program of China (grants nos. 2019YFC1200500 and 2019YFC1200502) and the Beijing Natural Science Foundation (grant no. 5204038) to Y.Z., the State Key Program of National Natural Science of China (grant no. U1808202) and NSFC International (regional) Cooperation and Exchange Program (grant no. 31961143024) to Z.C.

Author information

Authors and Affiliations



Y.F. conceived and supervised the project. Y.X., Z.G., P.Y., H.L. and H.W. purified the proteins, grew and optimized the crystals and performed the in vitro activity analysis and binding assays, supervised by Y.F. and Z.C. Y.F. collected the diffraction data and solved the crystal structure with the help of Y.Z. L.Z. collected the cryo-EM data and solved the cryo-EM structure, supervised by M.Y. Y.F. analyzed the data and wrote the manuscript, with assistance from all the authors.

Corresponding author

Correspondence to Yue Feng.

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The authors declare no competing interests.

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Nature Chemical Biology thanks Joseph Bondy-Denomy 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 AcrIF5 coelutes with the Csy-dsDNA complex but not the apo Csy.

Gel filtration profiles of the dsDNA and the mixes of Csy/dsDNA, Csy/AcrIF5 and Csy/dsDNA/AcrIF5. The UV absorbance at 280 nm is shown. The SDS-PAGE gels of the peak fractions of respective profile are shown on the right. The ‘*’ indicates contaminated proteins in the purified sample of the Csy complex. This experiment was performed 3 times with reproducible results.

Extended Data Fig. 2 Cryo-EM image processing for the Csy-dsDNA-AcrIF5 complex.

a, Representative processing pipeline includes a representative raw cryo-EM micrograph, 2D class averages, 3D classification and refinement of cryo-EM particles. b, Local resolution estimation and Euler angle distribution of the map of the Csy-dsDNA-AcrIF5 complex. c, Plot of the global FSC indicates an average resolution of 3.18 Å for the Csy-dsDNA-AcrIF5 complex.

Extended Data Fig. 3 Progressive displacement of Cas7f Subunits upon AcrIF5 binding to the Csy-dsDNA complex.

Comparison of each Cas7f subunit in Csy-dsDNA (PDB code: 6NE0, limon) and Csy-dsDNA-AcrIF5 (gray) complexes. The Cas7f subunits in the Csy-dsDNA complex become progressively displaced through mainly rigid-body movement as a result of AcrIF5 binding. Structures of the two complexes are aligned at Cas8f.

Extended Data Fig. 4 Structural superimposition of AcrIF5 with its structural homologs.

Structural superimposition of AcrIF5 with aPelota from Aeropyrum pernix K1 (PDB code: 6JI2, a), Cas12i2 (PDB code: 6LTP, b) and Tex protein from Pseudomonas aeruginosa (PDB code: 3BZC, c), respectively.

Extended Data Fig. 5 The crystal structure of AcrIF5.

a, Two AcrIF5 molecules are present in one asymmetric unit of the AcrIF5 crystal, colored in green and cyan. b, Structural superimposition of the two protomers of AcrIF5 in crystal. c, Structural superimposition of the two protomers of AcrIF5 in crystal (green and cyan) and the five AcrIF5 molecules in cryo-EM structure (colored as in Fig. 2a).

Extended Data Fig. 6 The interaction mode between AcrIF5 and Cas7f subunits.

a-d, Cartoon representation of the subcomplex of AcrIF5.4 (a), AcrIF5.3 (b), AcrIF5.2 (c), AcrIF5.1 (d), respectively. The web loops of the majorly bound Cas7f subunits are marked. e, Structural superimposition of subcomplexes of AcrIF5.5-5.1, which are shown in Fig. 3b and Extended Data Fig. 6a–d.

Extended Data Fig. 7 Structural mechanism of the binding specificity of AcrIF5 for the Csy-dsDNA complex.

a, Structural superimposition of the Cas7.2/7.3/7.4f subcomplex of the Csy (PDB code: 6B45) and Csy-dsDNA (PDB code: 6NE0), and the Cas7.2/7.3/7.4f-AcrIF5.3 subcomplex of the Csy-dsDNA-AcrIF5 (this study), aligned at the Cas7.2f subunit. b, Part of the structural superimposition in (a), in which only the Cas7.2/7.3/7.4f subcomplex of the Csy and AcrIF5.3 of the Cas7.2/7.3/7.4f-AcrIF5.3 subcomplex of the Csy-dsDNA-AcrIF5 are shown. Two clash regions are marked with circles. c, Part of the structural superimposition in (a), in which only the Cas7.2/7.3/7.4f subcomplex of the Csy-dsDNA and AcrIF5.3 of the Cas7.2/7.3/7.4f-AcrIF5.3 subcomplex of the Csy-dsDNA-AcrIF5 are shown. The corresponding regions of the two clash sites in (b) are marked with circles. d, On the basis of the structural superimposllition shown in (c), the Cas7.2f, 7.3f and 7.4f subunits in the structure of the Csy complex are aligned to their counterparts in the Csy-dsDNA structure. The same regions marked in (b) and (c) are also marked.

Extended Data Fig. 8 Structural superimposition of the Cas7f subunits of the Csy, Csy-dsDNA and Csy-dsDNA-AcrIF5 complexes.

a–f, Structural superimposition of the Cas7f subunits of the Csy (PDB code: 6B45, red), Csy-dsDNA (PDB code: 6NE0, limon) and Csy-dsDNA-AcrIF5 (this study, dark and light gray) complexes. The region of residues 232-243 of the web domain of Cas7.1f in the Csy-dsDNA-AcrIF5 complex is shown with cryo-EM density as blue mesh (a).

Extended Data Fig. 9 AcrIF5 coexists with AcrIF3 in the same operon.

Shown is a schematic representation of the genomic context of acrIF5 genes. Genes shown in the same color represent the same family. Genes encoding an HTH-containing protein, a phage virion morphogenesis protein and an SOS response-associated peptidase are colored green, gray and light pink, respectively. Arrows representing genes are not shown to scale.

Extended Data Fig. 10 MST binding assays of AcrIF5 and Cas2/3 to the Csy-dsDNA complex.

MST assay of the binding between the purified Csy-dsDNA complex and AcrIF5 (a) or Cas2/3 (b). The binding curves are displayed. Shown are mean values ± s.d.; n=3 biologically independent experiments. Individual data points are also shown.

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Xie, Y., Zhang, L., Gao, Z. et al. AcrIF5 specifically targets DNA-bound CRISPR-Cas surveillance complex for inhibition. Nat Chem Biol 18, 670–677 (2022).

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