Abstract
In biological systems, the activities of macromolecular complexes must sometimes be turned off. Thus, a wide variety of protein inhibitors has evolved for this purpose. These inhibitors function through diverse mechanisms, including steric blocking of crucial interactions, enzymatic modification of key residues or substrates, and perturbation of post-translational modifications1. Anti-CRISPRs—proteins that block the activity of CRISPR–Cas systems—are one of the largest groups of inhibitors described, with more than 90 families that function through diverse mechanisms2,3,4. Here, we characterize the anti-CRISPR AcrIF25, and we show that it inhibits the type I-F CRISPR–Cas system by pulling apart the fully assembled effector complex. AcrIF25 binds to the predominant CRISPR RNA-binding components of this complex, comprising six Cas7 subunits, and strips them from the RNA. Structural and biochemical studies indicate that AcrIF25 removes one Cas7 subunit at a time, starting at one end of the complex. Notably, this feat is achieved with no apparent enzymatic activity. To our knowledge, AcrIF25 is the first example of a protein that disassembles a large and stable macromolecular complex in the absence of an external energy source. As such, AcrIF25 establishes a paradigm for macromolecular complex inhibitors that may be used for biotechnological applications.
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Data availability
Unique identifiers and accession codes for the anti-CRISPR and anti-CRISPR-associated genes and their encoded proteins shown in Extended Data Fig. 1a are listed in Supplementary Table 1. Crystallography data and refinement statistics for Se-AcrIF25 and Cas7–AcrIF25 are reported in Extended Data Table 1. Atomic coordinates of the X-ray crystal structure models for Se-AcrIF25 and Cas7–AcrIF25 have been deposited at the PDB under accession codes 8JDH and 8JDI, respectively. The databases queried in this study are the NCBI Protein Database (https://www.ncbi.nlm.nih.gov/protein/) and the US Data Center for the global PDB (https://www.rcsb.org/pages/about-us/index). Three-dimensional structures generated by others that were used to support the data and conclusions in our study were taken from the PDB (5UZ9, 6B45 and 4AL5). The raw images corresponding to all figures are presented in Supplementary Fig. 1. An extensive list of the resources and regents used is reported in Supplementary Table 3.
Code availability
No custom computer code, algorithms or software were generated.
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Acknowledgements
We thank S. Stanley, A. Pawluk, B. Hicks and R. Wilson for their insights and discussions; M. Mejdani for technical contributions to this study; T. Moraes for advice on SEC–MALS; G. Wasney, J. M. Jorgensen, B. Cordeiro and M. Vargas for technical assistance with SEC–MALS experiments; B. Funnell and J. Baxter for advice on fluorescence spectroscopy; and the staff of the BL-17U1 and BL-19U1 beamlines at the Shanghai Synchrotron Radiation Facility. This work was supported by grants to Y.W. from the Natural Science Foundation of China (32330055, 31930065, 22121003), National Key R&D Program of China (2023YFC3403400, 2023YFA0915000), the Chinese Academy of Sciences (XDB0570000 and XDB37010202) and Beijing Municipal Science & Technology Commission (Z231100007223004); and grants to A.R.D. (MOP-130482 and FDN-15427) and K.L.M. (PJT-165936) from the Canadian Institutes of Health Research (CIHR). A.R.D. is the Canada Research Chair in Bacteriophage-Based Technologies (950-232058). Funding for this work was also provided to K.L.M. by the Natural Sciences and Engineering Research Council of Canada Arthur B. McDonald Fellowship (SMFSU-581368-2023). C.N.T. was supported by a CIHR Post-Doctoral Fellowship Award.
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Contributions
C.N.T., A.R.D. and K.L.M. conceptualized the study. Experimental design was guided by A.R.D., C.N.T., B.G., K.L.M. and Y.W. Bioinformatics was performed by C.N.T. Plasmids were constructed and propagated by C.N.T., W.-T.L., B.C.M.F., B.G., Y.H.-R., J.Y. and J.W. In vivo bacteriophage assays were performed by C.N.T., W.-T.L., B.C.M.F., B.G. and Y.H.-R. Biochemical characterizations of the proteins in this study were undertaken by C.N.T., W.-T.L., B.G. and Y.H.-R. under the supervision of A.R.D. and K.L.M. The X-ray crystal structure models for Se-AcrIF25 and Cas7–AcrIF25 were solved by J.Y. and J.W. under the supervision of Y.W. In vitro validation of the structure model for Cas7–AcrIF25 by mutational analysis was done by C.N.T., B.G. and Y.H.-R., and in vivo validation was performed by B.C.M.F., B.G. and Y.H.-R. All of the authors contributed to the manuscript by providing data, data interpretations, figures, methods and text. All of the authors read, reviewed and approved the final manuscript.
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A.R.D. is listed as an inventor on patents relating to anti-CRISPR proteins and is a scientific advisory board member for Acrigen Biosciences. The other authors declare no competing interests.
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Extended data figures and tables
Extended Data Fig. 1 Discovery of an anti-CRISPR protein, AcrIF25, that inhibits the type I-F CRISPR-Cas system of Pseudomonas aeruginosa.
a, Maps of genomic regions encoding Acrs and Acr-associated proteins (Acas) are shown. Horizontal arrows denote protein-encoding genes, horizontal lines represent intergenic regions, and a forward slash indicates the end of a contig. The type of mobile genetic element in which these regions lie is not clear. Red arrows indicate BLAST searches that were carried out to discover new Acr-encoding genes. These arrows begin at genes encoding a query protein and point to genes that were discovered. Acrs encoded by genes with checkmarks possess experimentally verified Acr activity40,41. b, In vivo assays of Acr activity against the endogenously expressed type I-F CRISPR-Cas system of P. aeruginosa (Pae) strain PA14 are shown. Tenfold serially diluted lysates of CRISPR-sensitive (DMS3m) and CRISPR-insensitive (DMS3) phages were spotted on lawns of P. aeruginosa cells expressing the indicated Acrs or truncated versions of AcrIF25 from plasmids. Cells expressing AcrIF8, a previously confirmed type I-F Acr14, were tested as a positive control. Phages were also tested on a ∆cas3 mutant, which lacks CRISPR-Cas activity. The left panel represents three biological replicates. The middle and right panels are representative of two biological replicates. Raw images are shown in Supplementary Fig. 1. c, Sequence alignment (MuscleWS) of AcrIF25 and its BLASTp matches (as of September 15, 2023) displayed using the Clustal colour scheme (Jalview). HHpred (MPI Bioinformatics Toolkit) searches revealed that three of these matches have the same two domains as AcrIF25: an N-terminal ribbon-helix-helix (RHH) domain and a C-terminal Acr domain. The fourth match to AcrIF25 has an N-terminal Acr domain and a C-terminal helix-turn-helix (HTH) domain. Percent identities (over a given alignment length) for all domains are reported. For the RHH and Acr domains, all comparisons were made relative to AcrIF25 (ARB47011.1). The percent identity for the HTH domain is reported relative to Aca1 (YP_007392343.1) of Pseudomonas phage JBD30 (ref. 14), because Aca1 is also a HTH domain protein. RHH domain elements are labelled as β1, ∝1, and ∝2. The residues on AcrIF25 that were mutated in the present study are indicated by asterisks, with blue asterisks indicating the three residues that showed the greatest effects when substituted with Ala. The end positions of AcrIF25 truncations that were tested in the middle and right-hand parts of panel b are indicated by black (N-terminal truncations) or red (C-terminal truncations) triangles, respectively. One triangle that is half black and half red marks the end position of both an N- and C-terminal truncation. Due to space limitations, some genera were abbreviated; full genus and species names are: Alloalcanivorax xenomutans, Marinobacter salarius, and Caldimonas thermodepolymerans.
Extended Data Fig. 2 The crystal structure of AcrIF25 and the AcrIF25:Cas7 complex.
a, The 2mFo-Fc electron density map (represented as mesh and contoured at 1.5 σ) of two regions of the Se-AcrIF25 structure (left two models; corresponding to PDB ID: 8JDH and represented by sticks), and the 2mFo-Fc electron density map (represented as mesh and contoured at 1.0 σ) of the same two regions of AcrIF25 in the structure of the Cas7:AcrIF25 complex (right two models; corresponding to PDB ID: 8JDI and represented by sticks). b, In the left panel, a cartoon representing the crystal structure of Apo-AcrIF25 (PDB ID: 8JDH) is shown. The two monomers of AcrIF25 are coloured aquamarine and pale yellow, and each monomer is comprised of an N-terminal RHH domain and a C-terminal Acr domain (CTD). In the right panel, a cartoon representation is shown of the crystal structure of AcrIF25 as it appears when bound to Cas7 (PDB ID: 8JDI). Cas7 is not shown here. The dimerized RHH domains are boxed to serve as a point of reference. c, A cartoon representation of the Cas7:AcrIF25 asymmetric unit is shown. The four Cas7 molecules (A, B, E, and G) are coloured blue. The two AcrIF25 molecules that are dimerized through interaction of their N-terminal RHH domains are coloured brown and pink (D and H, respectively). The RHH domain dimer is circled. The two other AcrIF25 molecules are coloured red (C and F), and each dimerizes with molecules in neighbouring asymmetric units. d, The Cas7 molecules (blue) found in the asymmetric unit of the Cas7:AcrIF25 crystal are depicted as ribbons and overlaid. Also overlaid and displayed as a ribbon is a Cas7 molecule (red) found within a Csy complex (PDB ID: 6B45; molecule F). Cas7 residues 55 to 75 were not detected in the Cas7:AcrIF25 structure. e, Cartoon representation of an AcrIF25 dimer from panel c (molecules D and H), coloured as in panel b, bound to two molecules of Cas7 (molecules A and G), also from panel c.
Extended Data Fig. 3 Effects of AcrIF25 mutants on Csy complex dissociation by size-exclusion chromatography.
Purified Csy complex containing Cas7 wild-type (WT) was incubated alone (a) or with a fourfold molar excess (relative to Cas7 subunits in the Csy complex) of purified WT or mutant AcrIF25 (b-g). Similarly, purified Csy complex containing Cas7 R50A was incubated alone (h) or with a fourfold molar excess of purified WT or mutant AcrIF25 (i-n). The resulting protein mixtures were fractionated by size-exclusion chromatography (SEC), and fractions were analysed by SDS-PAGE (top) and urea-PAGE (bottom). Input samples were loaded in the second lanes and representative fractions in lanes 3 to 15. Absorbance peaks (at 280 nm) are indicated. Peaks consistently observed upon mixing the WT Csy complex with WT AcrIF25 are numbered 1 to 3 as in Fig. 1c and d. The peak position of the intact Csy complex is also shown where relevant. The AcrIF25 mutant tested is indicated on the gel. Two biological replicates of these experiments were performed. The representative gels shown here are displayed in their entirety in Supplementary Fig. 1.
Extended Data Fig. 4 Analysis of AcrIF25 and Cas7 mutants.
The three AcrIF25 mutants that displayed reduced ability to dissociate wild-type (WT) Csy complex (Extended Data Fig. 3c, d, g) were purified by SEC. a, The elution profiles of WT and each mutant from the SEC column are overlaid. The colours of the traces correspond to the colours of the rectangles around the gels in b. The molecular weights (MWs) of the protein standards used to calibrate the S200 Increase 10/300 GL column are shown above the plot. The void volume (Vo), as measured by blue dextran, was 9.8 mL. b, Fractions from the SEC experiments shown in panel a were analysed by SDS-PAGE. Input samples were loaded in the second lanes and fractions in lanes 3–15 of each polyacrylamide gel. An arrow indicates the position of the absorbance peaks in each experiment. Each experiment was performed twice, as biological replicates, with a representative experiment shown. c, WT and mutant Csy complexes were purified by Ni-NTA affinity chromatography. The eluted fractions were analysed by SDS-PAGE (top) and urea-PAGE (bottom). Unlabelled bands on the SDS-PAGE gels are contaminants. Note that other Cas proteins and the crRNA did not co-purify with the Cas7 R150A mutant. The experiment shown is representative of two biological replicates. d, Phage replication assay with tenfold serial dilutions of phage Mu spotted onto lawns of E. coli expressing CRISPR-Cas complexes carrying different Cas7 mutants with and without AcrIF25. AcrIF25 was expressed in the absence of aTc. These experiments are representative of three biological replicates. See Supplementary Fig. 1 for the raw images corresponding to this figure.
Extended Data Fig. 5 Assessing the effect of ATP on AcrIF25 activity.
A limiting amount of AcrIF25 (AcrIF25 monomers:Cas7 monomers ratio = 0.3) was added to purified Csy complex and incubated for 15 min or 60 min. These reactions were carried out in the presence or absence of ATP and MgCl2. The reactions were terminated by Ni-NTA affinity chromatography and eluted samples were analysed by SDS-PAGE (top) and urea-PAGE (bottom). Reactions were also carried out with a fourfold excess of AcrIF25 to confirm that the AcrIF25 preparation was active. In another set of experiments designated as 120 min, a fourfold excess of AcrIF25 was preincubated in buffer for 60 min in the absence of the Csy complex and then it was added to Csy complex for an additional 60 min. This experiment demonstrated that AcrIF25 remained active for at least 60 min under the reaction conditions used. Identical reactions were carried out using AcrIF1 for each condition as a negative control. Molar ratios are expressed in terms of Acr monomers relative to the number of Cas7 molecules in the Csy complex. Two biological replicates of these experiments were performed with one representative experiment shown. Uncropped gel images are displayed in Supplementary Fig. 1.
Extended Data Fig. 6 Prediction of crRNA fragment sizes produced by RNase A digestion of the Csy complex.
The sequence of the crRNA component of the Csy complex used in the in vitro assays investigating AcrIF25 function is shown. The predicted RNase A cleavage sites in the crRNA are indicated by arrows. The numbering of these sites corresponds to the numbering shown in Fig. 5c–f. RNase A cleaves preferentially at 5′-YpR, where Y is C or U and R is A or G42. Sites marked by arrows match the RNase A consensus and cleavage at these sites would produce fragments that deviate by less than 2 nt compared to the measured sizes of the fragments produced in the experiment shown in Fig. 5c. The sizes of these fragments in nt were estimated using Image Lab (version 6.0.1, Bio-Rad) and two molecular markers: Low Range ssRNA Ladder and MicroRNA Marker (New England Biolabs). The experimentally measured and predicted RNase A cleavage fragment sizes are shown in the table. Fragment sizes were estimated by averaging the results from the experiments shown in Fig. 5c. Errors measures represent the standard error of the mean. Since RNase A will cleave any 5′-Yp site to some degree and samples were incubated for 45 min in an excess of RNase A, deviations between the predicted and measured fragment sizes can be accounted for by cleavage at 5′-YpY sites for fragments 1 and 2.
Extended Data Fig. 7 SEC analysis of Csy complex incubated with AcrIF1 and then AcrIF25.
a, Purified Csy complex was incubated with a fourfold molar excess of AcrIF1 followed by a fourfold molar excess of AcrIF25 monomers (relative to six Cas7 subunits in the Csy complex). The resulting mixture was fractionated by SEC. Absorbance measurements from the eluted fractions are plotted versus elution volume (red line). The elution profile of the intact Csy complex is also shown (black line). The peaks labelled 2 (Cas7:AcrIF25 complex) and 3 (AcrIF25 dimer) are the same as those observed when the Csy complex was mixed with excess AcrIF25 in the absence of AcrIF1 (Fig. 1c). However, peak 1 consistently eluted later than the intact Csy complex, but considerably earlier than peak 1 in Fig. 1c (complex of Cas8, Cas5, Cas6, and crRNA). The MW of the complex in peak 1 here was estimated to be 301 kDa, which is similar to the predicted MW of an AcrIF1-bound Csy complex with two Cas7 subunits removed (309 kDa). The MWs of the protein standards used to calibrate the S200 Increase 10/300 GL column are shown above the plot. b, Proteins present in the fractions from the SEC experiment described in panel a were analysed by SDS-PAGE (top). Fractions eluting at the peaks shown in panel a are indicated. The crRNA content of each fraction was detected by urea-PAGE. The portion of this gel where the crRNA migrated is shown beneath the SDS-PAGE gel. Representative results for three biological replicates are displayed here. Supplementary Fig. 1 contains the raw gel images.
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Trost, C.N., Yang, J., Garcia, B. et al. An anti-CRISPR that pulls apart a CRISPR–Cas complex. Nature 632, 375–382 (2024). https://doi.org/10.1038/s41586-024-07642-3
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DOI: https://doi.org/10.1038/s41586-024-07642-3
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