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Broad-spectrum anti-CRISPR proteins facilitate horizontal gene transfer

Nature Microbiology volume 5, pages 620–629 (2020)Cite this article

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Abstract

CRISPR–Cas adaptive immune systems protect bacteria and archaea against their invading genetic parasites, including bacteriophages/viruses and plasmids. In response to this immunity, many phages have anti-CRISPR (Acr) proteins that inhibit CRISPR–Cas targeting. To date, anti-CRISPR genes have primarily been discovered in phage or prophage genomes. Here, we uncovered acr loci on plasmids and other conjugative elements present in Firmicutes using the Listeria acrIIA1 gene as a marker. The four identified genes, found in Listeria, Enterococcus, Streptococcus and Staphylococcus genomes, can inhibit type II-A SpyCas9 or SauCas9, and are thus named acrIIA16–19. In Enterococcus faecalis, conjugation of a Cas9-targeted plasmid was enhanced by anti-CRISPRs derived from Enterococcus conjugative elements, highlighting a role for Acrs in the dissemination of plasmids. Reciprocal co-immunoprecipitation showed that each Acr protein interacts with Cas9, and Cas9–Acr complexes were unable to cleave DNA. Northern blotting suggests that these anti-CRISPRs manipulate single guide RNA length, loading or stability. Mirroring their activity in bacteria, AcrIIA16 and AcrIIA17 provide robust and highly potent broad-spectrum inhibition of distinct Cas9 proteins in human cells (for example, SpyCas9, SauCas9, SthCas9, NmeCas9 and CjeCas9). This work presents a focused analysis of non-phage Acr proteins, demonstrating a role in horizontal gene transfer bolstered by broad-spectrum CRISPR–Cas9 inhibition.

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Fig. 1: Identification of four type II-A Cas9 inhibitors, AcrIIA16–19.
Fig. 2: Prevalence of acrIIA genes in integrative MGEs and their effect on CRISPR targeting during conjugation.
Fig. 3: In vitro binding and inhibition activities of AcrIIA16–19 against SpyCas9.
Fig. 4: In vivo co-immunoprecipitation of AcrIIA16–19 and SpyCas9.
Fig. 5: Acr-mediated inhibition of Cas9 orthologues during gene editing in human cells.

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Data availability

All data generated or analysed during this study are included in this published article and its Extended Data, Source Data and Supplementary Information files. All relevant accession codes are available in Table 1.

Change history

  • 30 April 2020

    In the version of this Article originally published, Extended Data Fig. 4 was missing a label for panel c; this has now been corrected.

  • 23 April 2020

    A Correction to this paper has been published: https://doi.org/10.1038/s41564-020-0726-9

References

  1. Palmer, K. L., Kos, V. N. & Gilmore, M. S. Horizontal gene transfer and the genomics of enterococcal antibiotic resistance. Curr. Opin. Microbiol. 13, 632–639 (2010).

    Article  CAS  Google Scholar 

  2. Waldor, M. K. & Mekalanos, J. J. Lysogenic conversion by a filamentous phage encoding cholera toxin. Science 272, 1910–1914 (1996).

    Article  CAS  Google Scholar 

  3. Koonin, E. V. Viruses and mobile elements as drivers of evolutionary transitions. Philos. Trans. R. Soc. Lond. B 371, 20150442 (2016).

    Article  Google Scholar 

  4. Labrie, S. J., Samson, J. E. & Moineau, S. Bacteriophage resistance mechanisms. Nat. Rev. Microbiol. 8, 317–327 (2010).

    Article  CAS  Google Scholar 

  5. PriceV. J., Huo W., Sharifi A. & Palmer K. L. CRISPR-cas and restriction-modification act additively against conjugative antibiotic resistance plasmid transfer in enterococcus faecalis. mSphere 1, e00064-16 (2016).

    Article  Google Scholar 

  6. Edgar, R. & Qimron, U. The Escherichia coli CRISPR system protects from λ lysogenization, lysogens, and prophage induction. J. Bacteriol. 192, 6291–6294 (2010).

    Article  CAS  Google Scholar 

  7. Zhang, Y. et al. Processing-independent CRISPR RNAs limit natural transformation in Neisseria meningitidis. Mol. Cell 50, 488–503 (2013).

    Article  CAS  Google Scholar 

  8. Makarova, K. S. et al. An updated evolutionary classification of CRISPR–Cas systems. Nat. Rev. Microbiol. 13, 722–736 (2015).

    Article  CAS  Google Scholar 

  9. Garneau, J. E. et al. The CRISPR/cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature 468, 67–71 (2010).

    Article  CAS  Google Scholar 

  10. Clark, D. P. & Pazdernik, N. J. in Molecular Biology 2nd edn, Ch. e26 (Elsevier, 2013).

  11. Casjens, S. Prophages and bacterial genomics: what have we learned so far? Mol. Microbiol. 49, 277–300 (2003).

    Article  CAS  Google Scholar 

  12. Bondy-Denomy, J., Pawluk, A., Maxwell, K. L. & Davidson, A. R. Bacteriophage genes that inactivate the CRISPR/Cas bacterial immune system. Nature 493, 429–432 (2013).

    Article  CAS  Google Scholar 

  13. Zhu, Y. et al. Diverse mechanisms of CRISPR-Cas9 inhibition by type IIC Anti-CRISPR proteins. Mol. Cell 74, 296–309 (2019).

    Article  CAS  Google Scholar 

  14. Thavalingam, A. et al. Inhibition of CRISPR–Cas9 ribonucleoprotein complex assembly by anti-CRISPR AcrIIC2. Nat. Commun. 10, 2806 (2019).

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  16. Harrington, L. B. et al. A broad-spectrum inhibitor of CRISPR-Cas9. Cell 170, 1224–1233 (2017).

    Article  CAS  Google Scholar 

  17. Dong, L. et al. An anti-CRISPR protein disables type V Cas12a by acetylation. Nat. Struct. Mol. Biol. 26, 308–314 (2019).

    Article  CAS  Google Scholar 

  18. Knott, G. J. et al. Broad-spectrum enzymatic inhibition of CRISPR–Cas12a. Nat. Struct. Mol. Biol. 26, 315–321 (2019).

    Article  CAS  Google Scholar 

  19. Rauch, B. J. et al. Inhibition of CRISPR-Cas9 with bacteriophage proteins. Cell 168, 150–158 (2017).

    Article  CAS  Google Scholar 

  20. Hynes, A. P. et al. An anti-CRISPR from a virulent streptococcal phage inhibits Streptococcus pyogenes Cas9. Nat. Microbiol. 2, 1374–1380 (2017).

    Article  CAS  Google Scholar 

  21. Hynes, A. P. et al. Widespread anti-CRISPR proteins in virulent bacteriophages inhibit a range of Cas9 proteins. Nat. Commun. 9, 2919 (2018).

    Article  Google Scholar 

  22. Uribe, R. V. et al. Discovery and characterization of Cas9 inhibitors disseminated across seven bacterial phyla. Cell Host Microbe 25, 233–241 (2019).

    Article  CAS  Google Scholar 

  23. Forsberg, K. J. et al. Functional metagenomics-guided discovery of potent cas9 inhibitors in the human microbiome. eLife 8, e46540 (2019).

    Article  Google Scholar 

  24. Pawluk, A. et al. Naturally occurring off-switches for CRISPR-Cas9. Cell 167, 1829–1838 (2016).

    Article  CAS  Google Scholar 

  25. Jiang, F. et al. Temperature-responsive competitive inhibition of CRISPR-Cas9. Mol. Cell 73, 601–610 (2019).

    Article  CAS  Google Scholar 

  26. Liu, L., Yin, M., Wang, M. & Wang, Y. Phage AcrIIA2 DNA mimicry: structural basis of the CRISPR and anti-CRISPR arms race. Mol. Cell 73, 611–620 (2019).

    Article  CAS  Google Scholar 

  27. Dong, D. et al. Structural basis of CRISPR–SpyCas9 inhibition by an anti-CRISPR protein. Nature 546, 436–439 (2017).

    Article  CAS  Google Scholar 

  28. Shin, J. et al. Disabling Cas9 by an anti-CRISPR DNA mimic. Sci. Adv. 3, e1701620 (2017).

    Article  Google Scholar 

  29. Garcia, B. et al. Anti-CRISPR AcrIIA5 potently inhibits all Cas9 homologs used for genome editing. Cell Rep. 29, 1739–1746 (2019).

    Article  CAS  Google Scholar 

  30. Borges, A. L. et al. Bacteriophage cooperation suppresses CRISPR-Cas3 and Cas9 Immunity. Cell 174, 917–925 (2018).

    Article  CAS  Google Scholar 

  31. Osuna, B. A. et al. Listeria phages induce Cas9 degradation to protect lysogenic genomes. Preprint at https://doi.org/10.1101/787200 (2019).

  32. Hullahalli, K., Rodrigues, M. & Palmer, K. L. Exploiting CRISPR-Cas to manipulate Enterococcus faecalis populations. eLife 6, e26664 (2017).

    Article  Google Scholar 

  33. Trasanidou, D. et al. Keeping crispr in check: diverse mechanisms of phage-encoded anti-crisprs. FEMS Microbiol. Lett. 366, 1–14 (2019).

    Article  Google Scholar 

  34. Zhang, F., Song, G. & Tian, Y. Anti-CRISPRs: the natural inhibitors for CRISPR-Cas systems. Anim. Model. Exp. Med. 2, 69–75 (2019).

    Article  Google Scholar 

  35. PalmerK. L. & GilmoreM. S. Multidrug-resistant enterococci lack CRISPR-cas. mBio 1, e00227-10 (2010).

    Article  Google Scholar 

  36. Hullahalli, K., Rodrigues, M., Nguyen, U. T. & Palmer, K. An attenuated CRISPR-cas system in enterococcus faecalis permits DNA acquisition. mBio 9, e00414-18 (2018).

  37. Seamon, K. J., Light, Y. K., Saada, E. A., Schoeniger, J. S. & Harmon, B. Versatile high-throughput fluorescence assay for monitoring Cas9 activity. Anal. Chem. 90, 6913–6921 (2018).

    Article  CAS  Google Scholar 

  38. Hoang, T. T., Kutchma, A. J., Becher, A. & Schweizer, H. P. Integration-proficient plasmids for Pseudomonas aeruginosa: site-specific integration and use for engineering of reporter and expression strains. Plasmid 43, 59–72 (2000).

    Article  CAS  Google Scholar 

  39. Choi, K. H., Kumar, A. & Schweizer, H. P. A 10-min method for preparation of highly electrocompetent Pseudomonas aeruginosa cells: application for DNA fragment transfer between chromosomes and plasmid transformation. J. Microbiol. Methods 64, 391–397 (2006).

    Article  CAS  Google Scholar 

  40. Bhardwaj, P., Ziegler, E. & Palmer, K. L. Chlorhexidine induces VanA-type Vancomycin resistance genes in enterococci. Antimicrob. Agents Chemother. 60, 2209–2221 (2016).

    Article  CAS  Google Scholar 

  41. Kleinstiver, B. P. et al. Engineered CRISPR–Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing. Nat. Biotechnol. 37, 276–282 (2019).

    Article  CAS  Google Scholar 

  42. Clement, K. et al. CRISPResso2 provides accurate and rapid genome editing sequence analysis. Nat. Biotechnol. 37, 224–226 (2019).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank K. L. Palmer (UT Dallas) and G. M. Dunny (University of Minnesota) for providing all E. faecalis strains, vector and protocols. We thank M. Johnson for his assistance in executing PlasFlow, N. Marino for her profound wisdom and comedic wit, and all members of the Bondy-Denomy Lab for their continuous support and thoughtful discussions contributing to this study. Bondy-Denomy Lab was supported by the UCSF Program for Breakthrough Biomedical Research funded in part by the Sandler Foundation, the Searle Fellowship, the Vallee Foundation, an NIH Director’s Early Independence Award No. DP5-OD021344, and NIH grant no. R01GM127489. Research in the Kleinstiver Lab is supported by NIH grant no. R00-CA218870, an A.S.G.C.T. Career Development Award and the Margaret Q. Landenberger Research Foundation. This research was carried out using Defense Advanced Research Projects Agency (DARPA) award HR0011-17-2-0043. The views, opinions and/or findings should be attributed to the author and should not be interpreted as representing the official views or policies of the Department of Defense or the US Government.

Author information

Authors and Affiliations

  1. Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA, USA

    Caroline Mahendra, Beatriz A. Osuna, Rafael Pinilla-Redondo & Joseph Bondy-Denomy

  2. Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, USA

    Kathleen A. Christie & Benjamin P. Kleinstiver

  3. Department of Pathology, Massachusetts General Hospital, Boston, MA, USA

    Kathleen A. Christie & Benjamin P. Kleinstiver

  4. Department of Pathology, Harvard Medical School, Boston, MA, USA

    Kathleen A. Christie & Benjamin P. Kleinstiver

  5. Section of Microbiology, University of Copenhagen, Universitetsparken 15, Copenhagen, Denmark

    Rafael Pinilla-Redondo

  6. Department of Technological Educations, University College Copenhagen, Sigurdsgade 26, Copenhagen, Denmark

    Rafael Pinilla-Redondo

  7. Quantitative Biosciences Institute, University of California, San Francisco, San Francisco, CA, USA

    Joseph Bondy-Denomy

  8. Innovative Genomics Institute, Berkeley, CA, USA

    Joseph Bondy-Denomy

Authors
  1. Caroline Mahendra
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Contributions

C.M. and J.B.-D. conceived and designed the study. C.M. conducted bioinformatic, bacterial and biochemical experiments. K.A.C. performed human cell experiments. B.A.O. and R.P.-R. assisted with experimental design. B.P.K and J.B.-D. supervised experiments. C.M. and J.B.-D. wrote the manuscript with input from all authors.

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Correspondence to Joseph Bondy-Denomy.

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Competing interests

J.B.-D. is a scientific advisory board member of SNIPR Biome and Excision Biotherapeutics, and a scientific advisory board member and co-founder of Acrigen Biosciences. B.P.K. is an inventor on various patents and patent applications that describe gene editing and epigenetic editing technologies, is a consultant for Avectas Inc. and an advisor for Acrigen Biosciences.

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Extended data

Extended Data Fig. 1 Schematic of acr loci and lethal self-genome cleavage assay.

a, Full schematic of acr loci with relevant neighboring genes displayed. b, Schematic of SpyCas9 in P. aeruginosa programmed to cause lethal self-genome cleavage to assess bacterial survival in the presence of AcrIIA proteins. CRISPR strength is determined by titrating levels of IPTG, which induces expression of sgRNA targeting the chromosomal phzM gene from a multicopy plasmid. OD600 measurements are represented as the mean of three biological replicates ± SD.

Extended Data Fig. 2 Anti-CRISPR distribution in integrative mobile genetic elements across bacterial taxa.

Phylogenetic analysis based on acrIIA16–19 homologs (panels a to d, respectively) reconstructed from a midpoint rooted minimum-evolution of full length protein sequences identified following an iterative PSI-BLASTp search, see methods for details. Number of genomes included to construct each tree for acrIIA16–19 are seventy, twenty-six, eighty-four and seventeen respectively. Branches are labeled with species name and colored according to species class (see legend). Species for which AcrIIA homologs have been tested in this study are shown in bold.

Extended Data Fig. 3 AcrIIA enhance conjugation-mediated horizontal gene transfer in E. faecalis, related to Fig. 2.

a, b. Mating outcomes during plasmid conjugation of a targeted plasmid from donor to recipient cells where indicated acrIIA genes are (a) pre-expressed in recipient cells, or (b) encoded on conjugating plasmid. Data displayed as 10-fold colony serial dilution spots of donor, recipient or transconjugant cells on selective antibiotic plates. Mating assays were performed in biological triplicate and produced similar outcomes. c, Schematic of E. faecalis conjugation of protospacer and acrIIA-bearing plasmid transferring into CRISPR-defective recipients. For CRISPR1, the bona fide AcrIIA4 is utilized to suppress CRISPR targeting, and a ΔCas9 strain from previously reported work is used for CRISPR3 (Price et al.,5). Red * denotes plasmids that have lost conjugation ability. Mating assays were performed in biological duplicate or triplicate and produced similar outcomes.

Extended Data Fig. 4 AcrIIA16–19 biochemical analysis, related to Figs. 3 and 4.

a, Coomassie-stained polyacrylamide gel showing GST-tagged AcrIIA proteins (IIA4 37kD, IIA16Lmo 50kD, IIA17Sga 39kD, IIA18Sma 48kD and IIA19Ssim 42kD) purified from E. coli by elution from Glutathione Sepharose columns. Visible bands at different sizes are co-purifying proteins from E. coli. Data shown are representative of two independent experiments. b, Coomassie-stained polyacrylamide gel showing co-immunoprecipitation of Acr proteins with Myc-tagged sgRNA-bound SpyCas9 pulled down from P. aeruginosa. Data shown are representative of two independent experiments. c, d, Uncropped versions of both Myc and GST pulldowns from Fig. 4a and c, displaying all fragments of (c) sgRNA-bound SpyCas9, or (d) Apo- SpyCas9 without sgRNA present. Data shown are representative of two independent experiments.

Source data

Extended Data Fig. 5 Acr inhibition activity in human cells tested against different Cas9 orthologs, related to Fig. 5.

Reported Acr proteins in this study and from previous works tested for inhibition of genome editing activities of Sth1Cas9, Sth3Cas9, Nme2Cas9 and CjeCas9 (a-d, respectively). Editing efficiencies against endogenous genes in HEK 293 T cells were assessed by targeted sequencing and quantified as the percentage of reads containing a nuclease-induced alteration; the no-Acr condition contains an EGFP expression plasmid; the NT control includes an empty U6 expression plasmid. Percent reads modified are represented as the mean of three biological replicates ± SD.

Source data

Supplementary information

Supplementary Information

Supplementary Information for Extended Data 2 phylogenetic tree analyses. Raw versions of the trees in NEXUS format.

Reporting Summary

Supplementary Tables

Supplementary Tables 1–4.

Source data

Source Data Fig. 1

Statistical source data.

Source Data Fig. 2

Statistical source data.

Source Data Fig. 3

Unprocessed gels and northern blots.

Source Data Fig. 4

Unprocessed gels and western blots.

Source Data Fig. 5

Statistical source data.

Source Data Extended Data Fig. 4

Unprocessed gels and western blots.

Source Data Extended Data Fig. 5

Statistical source data.

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Mahendra, C., Christie, K.A., Osuna, B.A. et al. Broad-spectrum anti-CRISPR proteins facilitate horizontal gene transfer. Nat Microbiol 5, 620–629 (2020). https://doi.org/10.1038/s41564-020-0692-2

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