Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

CRISPR-repressed toxin–antitoxin provides herd immunity against anti-CRISPR elements

Nature Chemical Biology volume 21, pages 337–347 (2025)Cite this article

Subjects

Abstract

Prokaryotic clustered regularly interspaced short palindromic repeat (CRISPR)–Cas systems are highly vulnerable to phage-encoded anti-CRISPR (Acr) factors. How CRISPR–Cas systems protect themselves remains unclear. Here we uncovered a broad-spectrum anti-anti-CRISPR strategy involving a phage-derived toxic protein. Transcription of this toxin is normally repressed by the CRISPR–Cas effector but is activated to halt cell division when the effector is inhibited by any anti-CRISPR proteins or RNAs. We showed that this abortive infection-like effect efficiently expels Acr elements from bacterial population. Furthermore, we exploited this anti-anti-CRISPR mechanism to develop a screening method for specific Acr candidates for a CRISPR–Cas system and successfully identified two distinct Acr proteins that enhance the binding of CRISPR effector to nontarget DNA. Our data highlight the broad-spectrum role of CRISPR-repressed toxins in counteracting various types of Acr factors. We propose that the regulatory function of CRISPR–Cas confers host cells herd immunity against Acr-encoding genetic invaders whether they are CRISPR targeted or not.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to the full article PDF.

USD 39.95

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: The ADP1 CRISPR–Cas encodes a bacteriostatic protein (CreP) that induces cell filamentation.
Fig. 2: Characterization of CreA sequence and its inhibition of PcreP.
Fig. 3: CreP toxicity can be triggered by various Acr proteins and Racr RNAs.
Fig. 4: CrePA can be used for screening new Acr candidates.
Fig. 5: Both AcrIF25 and AcrIF26 induce the binding of Csy complex to nontarget DNA.
Fig. 6: Stability of Acr-expressing plasmids in ADP1 cell population.

Similar content being viewed by others

Data availability

All relevant data are included in the article and/or Supplementary Information. The coordinates and structure factors for AcrIF25 were deposited to the PDB under accession number 8X24. The raw data for the RNAseq experiments in Fig. 2 were deposited to the NCBI with the BioProject accession number PRJNA1100312. All strains and plasmids are available from the corresponding author upon request; requests will be answered within 2 weeks. Source data are provided with this paper.

References

  1. Barrangou, R. & Horvath, P. A decade of discovery: CRISPR functions and applications. Nat. Microbiol. 2, 17092 (2017).

    Article  CAS  PubMed  Google Scholar 

  2. Wiedenheft, B., Sternberg, S. H. & Doudna, J. A. RNA-guided genetic silencing systems in bacteria and archaea. Nature 482, 331–338 (2012).

    Article  CAS  PubMed  Google Scholar 

  3. Hille, F. et al. The biology of CRISPR–Cas: backward and forward. Cell 172, 1239–1259 (2018).

    Article  CAS  PubMed  Google Scholar 

  4. Nussenzweig, P. M. & Marraffini, L. A. Molecular mechanisms of CRISPR–Cas immunity in bacteria. Annu. Rev. Genet. 54, 93–120 (2020).

    Article  CAS  PubMed  Google Scholar 

  5. Jackson, S. A. et al. CRISPR–Cas: adapting to change. Science 356, eaal5056 (2017).

    Article  PubMed  Google Scholar 

  6. Makarova, K. S. et al. Evolutionary classification of CRISPR–Cas systems: a burst of class 2 and derived variants. Nat. Rev. Microbiol. 18, 67–83 (2020).

    Article  CAS  PubMed  Google Scholar 

  7. Sternberg, S. H., Richter, H., Charpentier, E. & Qimron, U. Adaptation in CRISPR–Cas systems. Mol. Cell 61, 797–808 (2016).

    Article  CAS  PubMed  Google Scholar 

  8. Semenova, E. et al. Interference by clustered regularly interspaced short palindromic repeat (CRISPR) RNA is governed by a seed sequence. Proc. Natl Acad. Sci. USA 108, 10098–10103 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Wiedenheft, B. et al. RNA-guided complex from a bacterial immune system enhances target recognition through seed sequence interactions. Proc. Natl Acad. Sci. USA 108, 10092–10097 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Bondy-Denomy, J. et al. Multiple mechanisms for CRISPR–Cas inhibition by anti-CRISPR proteins. Nature 526, 136–139 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Borges, A. L., Davidson, A. R. & Bondy-Denomy, J. The discovery, mechanisms, and evolutionary impact of anti-CRISPRs. Annu. Rev. Virol. 4, 37–59 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Jia, N. & Patel, D. J. Structure-based functional mechanisms and biotechnology applications of anti-CRISPR proteins. Nat. Rev. Mol. Cell Biol. 22, 563–579 (2021).

    Article  CAS  PubMed  Google Scholar 

  13. Yin, P., Zhang, Y., Yang, L. & Feng, Y. Non-canonical inhibition strategies and structural basis of anti-CRISPR proteins targeting type I CRISPR–Cas systems. J. Mol. Biol. 435, 167996 (2023).

    Article  CAS  PubMed  Google Scholar 

  14. Camara-Wilpert, S. et al. Bacteriophages suppress CRISPR–Cas immunity using RNA-based anti-CRISPRs. Nature 623, 601–607 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Faure, G. et al. CRISPR–Cas in mobile genetic elements: counter-defence and beyond. Nat. Rev. Microbiol. 17, 513–525 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Li, M. et al. Toxin–antitoxin RNA pairs safeguard CRISPR–Cas systems. Science 372, eabe5601 (2021).

    Article  CAS  PubMed  Google Scholar 

  17. Liu, C. et al. Widespread RNA-based cas regulation monitors crRNA abundance and anti-CRISPR proteins. Cell Host Microbe 31, 1481–1493 (2023).

    Article  CAS  PubMed  Google Scholar 

  18. Shmakov, S. A. et al. Widespread CRISPR-derived RNA regulatory elements in CRISPR–Cas systems. Nucleic Acids Res. 51, 8150–8168 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Workman, R. et al. A natural single-guide RNA repurposes Cas9 to autoregulate CRISPR–Cas expression. Cell 184, 675–688 (2021).

    Article  CAS  PubMed  Google Scholar 

  20. Ratner, H. K. et al. Catalytically active Cas9 mediates transcriptional interference to facilitate bacterial virulence. Mol. Cell 75, 498–510 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Cheng, F. et al. Divergent degeneration of creA antitoxin genes from minimal CRISPRs and the convergent strategy of tRNA-sequestering CreT toxins. Nucleic Acids Res. 49, 10677–10688 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Wang, R. et al. Associate toxin–antitoxin with CRISPR–Cas to kill multidrug-resistant pathogens. Nat. Commun. 14, 2078 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Richter, C. et al. Priming in the type IF CRISPR–Cas system triggers strand-independent spacer acquisition, bi-directionally from the primed protospacer. Nucleic Acids Res. 42, 8516–8526 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Iyer, L. M., Koonin, E. V. & Aravind, L. Extensive domain shuffling in transcription regulators of DNA viruses and implications for the origin of fungal APSES transcription factors. Genome Biol. 3, 1–11 (2002).

    Article  Google Scholar 

  25. Hansen, E. B. Structure and regulation of the lytic replicon of phage P1. J. Mol. Biol. 207, 135–149 (1989).

    Article  CAS  PubMed  Google Scholar 

  26. Das, A., Biswas, S. & Biswas, M. Expression of Phi11 Gp07 causes filamentation in Escherichia coli. Open Microbiol. J. 12, 107 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Cech, G. M., Szalewska-Pałasz, A., Potrykus, K. & Kloska, A. Virus–host interaction gets curiouser and curiouser. Part II: functional transcriptomics of the E. coli DksA-deficient cell upon phage P1 vir infection. Int. J. Mol. Sci. 22, 6159 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. de Boer, P. A. J. Advances in understanding E. coli cell fission. Curr. Opin. Microbiol. 13, 730–737 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  29. Richter, C., Gristwood, T., Clulow, J. S. & Fineran, P. C. In vivo protein interactions and complex formation in the Pectobacterium atrosepticum subtype IF CRISPR/Cas system. PLoS ONE 7, e49549 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Cheng, F. et al. The toxin–antitoxin RNA guards of CRISPR–Cas evolved high specificity through repeat degeneration. Nucleic Acids Res. 50, 9442–9452 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  32. Gao, Z. et al. Anti-CRISPR protein AcrIF4 inhibits the type IF CRISPR–Cas surveillance complex by blocking nuclease recruitment and DNA cleavage. J. Biol. Chem. 298, 102575 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Yang, L. et al. Insights into the inhibition of type IF CRISPR–Cas system by a multifunctional anti-CRISPR protein AcrIF24. Nat. Commun. 13, 1931 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Mukherjee, I. A., Gabel, C., Noinaj, N., Bondy-Denomy, J. & Chang, L. Structural basis of AcrIF24 as an anti-CRISPR protein and transcriptional suppressor. Nat. Chem. Biol. 18, 1417–1424 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Wu, S. et al. A pyrF-based efficient genetic manipulation platform in Acinetobacter baumannii to explore the vital DNA components of adaptive immunity for IF CRISPR–Cas. Microbiol. Spectr. 10, e01957-22 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  36. Jia, J. et al. Post-transcriptional regulation is involved in the cold-active methanol-based methanogenic pathway of a psychrophilic methanogen. Environ. Microbiol. 23, 3773–3788 (2021).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by the Science and Technology Fundamental Resources Investigation Program (2022FY101100 to H.Z.), the National Natural Science Foundation of China (32150020 to M.L., 82225028 to S.O., 32270092 to R.W., 32022003 to M.L., 32370090 to H.Z., 32200057 to F.C. and 82172287 to S.O.), the National Key Research and Development Program of China (2021YFC2301403 to S.O.), the Youth Innovation Promotion Association of the Chinese Academy of Sciences (2020090 to M.L.), the China National Postdoctoral Program for Innovative Talents (BX20220331 to F.C.), the China Postdoctoral Science Foundation (2022M720160 to F.C.) and the Special Research Assistant Program of the Chinese Academy of Sciences (2023000056 to F.C.).

Author information

Author notes

  1. These authors contributed equally: Xian Shu, Rui Wang, Zhihua Li, Qiong Xue.

Authors and Affiliations

  1. Department of Microbial Physiological & Metabolic Engineering, State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China

    Xian Shu, Rui Wang, Zhihua Li, Qiong Xue, Feiyue Cheng, Chao Liu, Huiwei Zhao & Ming Li

  2. College of Life Science, University of Chinese Academy of Sciences, Beijing, China

    Zhihua Li

  3. Key Laboratory of Microbial Pathogenesis and Interventions of Fujian Province University, Key Laboratory of Innate Immune Biology of Fujian Province, Biomedical Research Center of South China, Key Laboratory of Optoelectronic Science and Technology for Medicine of the Ministry of Education, College of Life Sciences, Fujian Normal University, Fuzhou, China

    Jiajun Wang & Songying Ouyang

  4. Institutional Center for Shared Technologies and Facilities of Institute of Microbiology, Chinese Academy of Sciences, Beijing, China

    Jingfang Liu

  5. Department of Biological Sciences, Faculty of Science, Department of Biochemistry, Yong Loo Lin School of Medicine, Precision Medicine Translational Research Programme (TRP), National University of Singapore, Singapore, Singapore

    Chunyi Hu

  6. State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China

    Jie Li & Ming Li

Authors
  1. Xian Shu
    View author publications

    Search author on:PubMed Google Scholar

  2. Rui Wang
    View author publications

    Search author on:PubMed Google Scholar

  3. Zhihua Li
    View author publications

    Search author on:PubMed Google Scholar

  4. Qiong Xue
    View author publications

    Search author on:PubMed Google Scholar

  5. Jiajun Wang
    View author publications

    Search author on:PubMed Google Scholar

  6. Jingfang Liu
    View author publications

    Search author on:PubMed Google Scholar

  7. Feiyue Cheng
    View author publications

    Search author on:PubMed Google Scholar

  8. Chao Liu
    View author publications

    Search author on:PubMed Google Scholar

  9. Huiwei Zhao
    View author publications

    Search author on:PubMed Google Scholar

  10. Chunyi Hu
    View author publications

    Search author on:PubMed Google Scholar

  11. Jie Li
    View author publications

    Search author on:PubMed Google Scholar

  12. Songying Ouyang
    View author publications

    Search author on:PubMed Google Scholar

  13. Ming Li
    View author publications

    Search author on:PubMed Google Scholar

Contributions

M.L. and R.W. conceptualized this study. M.L., R.W., X.S., J. Li and S.O. designed the experiments, with valuable suggestions from C.H. X.S., R.W. and Q.X. constructed the plasmids and mutant strains with the assistance of F.C., C.L. and H.Z. X.S. and R.W. conducted the dilution plating assay, SEM investigation, fluorescence measurement and bacteria transformation assays. X.S., R.W. and H.Z. performed RNAseq and primer extension. R.W., X.S. and Q.X. performed the bioinformatic analyses of CrePA and Acrs. Z.L. and X.S. conducted protein purification, western blot, SEC, TAP and EMSAs with the assistance of J. Li and J. Liu. X.S. conducted the DNA competition experiments and cell population assays. J.W. conducted crystallization and X-ray diffraction data collection of AcrIF25. Formal analysis of the results was performed by X.S., R.W., Z.L., Q.X., J. Li and S.O. M.L., J. Li, S.O. and R.W. analyzed the data and supervised the project. M.L. wrote the paper, which was edited and approved by all authors.

Corresponding authors

Correspondence to Rui Wang, Jie Li, Songying Ouyang or Ming Li.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Chemical Biology thanks Jennifer Doudna, Yue Feng and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information (download PDF )

Supplementary Table 1 and Figs. 1–10.

Reporting Summary (download PDF )

Supplementary Data 1 (download XLSX )

A. baylyi ADP1 crePA and its homologs.

Supplementary Data 2 (download XLSX )

Strains, chemicals, plasmids, oligonucleotides and synthetic genes.

Supplementary Data 3 (download XLSX )

Source data for supplementary figures.

Source data

Source Data Fig. 1 (download XLSX )

Statistical source data.

Source Data Fig. 2 (download XLSX )

Statistical source data.

Source Data Fig. 3 (download XLSX )

Statistical source data.

Source Data Fig. 4 (download XLSX )

Statistical source data.

Source Data Fig. 5 (download PDF )

Statistical source data.

Source Data Fig. 6 (download XLSX )

Statistical source data.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Shu, X., Wang, R., Li, Z. et al. CRISPR-repressed toxin–antitoxin provides herd immunity against anti-CRISPR elements. Nat Chem Biol 21, 337–347 (2025). https://doi.org/10.1038/s41589-024-01693-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Version of record:

  • Issue date:

  • DOI: https://doi.org/10.1038/s41589-024-01693-3

This article is cited by

Search

Advanced search

Quick links

Nature Briefing Microbiology

Sign up for the Nature Briefing: Microbiology newsletter — what matters in microbiology research, free to your inbox weekly.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing: Microbiology