Recombination between phages and CRISPR−cas loci facilitates horizontal gene transfer in staphylococci


CRISPR (clustered regularly interspaced short palindromic repeats) loci and their associated (cas) genes encode an adaptive immune system that protects prokaryotes from viral1 and plasmid2 invaders. Following viral (phage) infection, a small fraction of the prokaryotic cells are able to integrate a small sequence of the invader’s genome into the CRISPR array1. These sequences, known as spacers, are transcribed and processed into small CRISPR RNA guides3,4,5 that associate with Cas nucleases to specify a viral target for destruction6,7,8,9. Although CRISPR−cas loci are widely distributed throughout microbial genomes and often display hallmarks of horizontal gene transfer10,11,12, the drivers of CRISPR dissemination remain unclear. Here, we show that spacers can recombine with phage target sequences to mediate a form of specialized transduction of CRISPR elements. Phage targets in phage 85, ΦNM1, ΦNM4 and Φ12 can recombine with spacers in either chromosomal or plasmid-borne CRISPR loci in Staphylococcus, leading to either the transfer of CRISPR-adjacent genes or the propagation of acquired immunity to other bacteria in the population, respectively. Our data demonstrate that spacer sequences not only specify the targets of Cas nucleases but also can promote horizontal gene transfer.

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Fig. 1: Transfer of CRISPR–Cas elements through spacer-mediated transduction.
Fig. 2: Spacer sequences determine the frequency of pCRISPR transduction.
Fig. 3: Spacers that mediate high pCRISPR transduction provide poor immunity to the host.

Code availability

All codes used in this study are available on request from the corresponding author.

Data availability

All data generated or analysed during this study are included in this published article (and its Supplementary Information files). Raw sequencing data are available on request from the corresponding author.


  1. 1.

    Barrangou, R. et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science 315, 1709–1712 (2007).

    CAS  Article  Google Scholar 

  2. 2.

    Marraffini, L. A. & Sontheimer, E. J. CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA. Science 322, 1843–1845 (2008).

    CAS  Article  Google Scholar 

  3. 3.

    Brouns, S. J. et al. Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 321, 960–964 (2008).

    CAS  Article  Google Scholar 

  4. 4.

    Carte, J., Wang, R., Li, H., Terns, R. M. & Terns, M. P. Cas6 is an endoribonuclease that generates guide RNAs for invader defense in prokaryotes. Genes Dev. 22, 3489–3496 (2008).

    CAS  Article  Google Scholar 

  5. 5.

    Deltcheva, E. et al. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 471, 602–607 (2011).

    CAS  Article  Google Scholar 

  6. 6.

    Jore, M. M. et al. Structural basis for CRISPR RNA-guided DNA recognition by Cascade. Nat. Struct. Mol. Biol. 18, 529–536 (2011).

    CAS  Article  Google Scholar 

  7. 7.

    Samai, P. et al. Co-transcriptional DNA and RNA cleavage during type III CRISPR–Cas immunity. Cell 161, 1164–1174 (2015).

    CAS  Article  Google Scholar 

  8. 8.

    Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).

    CAS  Article  Google Scholar 

  9. 9.

    Gasiunas, G., Barrangou, R., Horvath, P. & Siksnys, V. Cas9–crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc. Natl Acad. Sci. USA 109, E2579–E2586 (2012).

    CAS  Article  Google Scholar 

  10. 10.

    Makarova, K. S., Aravind, L., Grishin, N. V., Rogozin, I. B. & Koonin, E. V. A DNA repair system specific for thermophilic archaea and bacteria predicted by genomic context analysis. Nucleic Acids Res. 30, 482–496 (2002).

    CAS  Article  Google Scholar 

  11. 11.

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

    CAS  Article  Google Scholar 

  12. 12.

    Jansen, R., Embden, J. D., Gaastra, W. & Schouls, L. M. Identification of genes that are associated with DNA repeats in prokaryotes. Mol. Microbiol. 43, 1565–1575 (2002).

    CAS  Article  Google Scholar 

  13. 13.

    Shmakov, S. A., Makarova, K. S., Wolf, Y. I., Severinov, K. V. & Koonin, E. V. Systematic prediction of genes functionally linked to CRISPR–Cas systems by gene neighborhood analysis. Proc. Natl Acad. Sci. USA 115, E5307–E5316 (2018).

    CAS  Article  Google Scholar 

  14. 14.

    Koonin, E. V. & Makarova, K. S. Mobile genetic elements and evolution of CRISPR–Cas systems: all the way there and back. Genome Biol. Evol. 9, 2812–2825 (2017).

    CAS  Article  Google Scholar 

  15. 15.

    Westra, E. R., Dowling, A. J., Broniewski, J. M. & van Houte, S. Evolution and ecology of CRISPR. Annu. Rev. Ecol. Evol. Syst. 47, 307–331 (2016).

    Article  Google Scholar 

  16. 16.

    Haft, D. H., Selengut, J., Mongodin, E. F. & Nelson, K. E. A guild of 45 CRISPR-associated (Cas) protein families and multiple CRISPR/Cas subtypes exist in prokaryotic genomes. PLoS Comput. Biol. 1, e60 (2005).

    Article  Google Scholar 

  17. 17.

    Chakraborty, S. et al. Comparative network clustering of direct repeats (DRs) and cas genes confirms the possibility of the horizontal transfer of CRISPR locus among bacteria. Mol. Phylogenet. Evol. 56, 878–887 (2010).

    CAS  Article  Google Scholar 

  18. 18.

    Godde, J. S. & Bickerton, A. The repetitive DNA elements called CRISPRs and their associated genes: evidence of horizontal transfer among prokaryotes. J. Mol. Evol. 62, 718–729 (2006).

    CAS  Article  Google Scholar 

  19. 19.

    Millen, A. M., Horvath, P., Boyaval, P. & Romero, D. A. Mobile CRISPR/Cas-mediated bacteriophage resistance in Lactococcus lactis. PLoS ONE 7, e51663 (2012).

    CAS  Article  Google Scholar 

  20. 20.

    Zinder, N. D. & Lederberg, J. Genetic exchange in Salmonella. J. Bacteriol. 64, 679–699 (1952).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Thomas, C. M. & Nielsen, K. M. Mechanisms of, and barriers to, horizontal gene transfer between bacteria. Nat. Rev. Microbiol. 3, 711–721 (2005).

    CAS  Article  Google Scholar 

  22. 22.

    Watson, B. N. J., Staals, R. H. J. & Fineran, P. C. CRISPR–Cas-mediated phage resistance enhances horizontal gene transfer by transduction. mBio 9, e02406-17 (2018).

    Article  Google Scholar 

  23. 23.

    Touchon, M., Moura de Sousa, J. A. & Rocha, E. P. Embracing the enemy: the diversification of microbial gene repertoires by phage-mediated horizontal gene transfer. Curr. Opin. Microbiol. 38, 66–73 (2017).

    CAS  Article  Google Scholar 

  24. 24.

    Orbach, M. J. & Jackson, E. N. Transfer of chimeric plasmids among Salmonella typhimurium strains by P22 transduction. J. Bacteriol. 149, 985–994 (1982).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Deichelbohrer, I., Alonso, J. C., Luder, G. & Trautner, T. A. Plasmid transduction by Bacillus subtilis bacteriophage SPP1: effects of DNA homology between plasmid and bacteriophage. J. Bacteriol. 162, 1238–1243 (1985).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Novick, R. P., Edelman, I. & Lofdahl, S. Small Staphylococcus aureus plasmids are transduced as linear multimers that are formed and resolved by replicative processes. J. Mol. Biol. 192, 209–220 (1986).

    CAS  Article  Google Scholar 

  27. 27.

    Maniv, I., Jiang, W., Bikard, D. & Marraffini, L. A. Impact of different target sequences on type III CRISPR–Cas immunity. J. Bacteriol. 198, 941–950 (2016).

    CAS  Article  Google Scholar 

  28. 28.

    Golding, G. R. et al. Whole-genome sequence of livestock-associated ST398 methicillin-resistant Staphylococcus aureus isolated from humans in Canada. J. Bacteriol. 194, 6627–6628 (2012).

    CAS  Article  Google Scholar 

  29. 29.

    Kwan, T., Liu, J., DuBow, M., Gros, P. & Pelletier, J. The complete genomes and proteomes of 27 Staphylococcus aureus bacteriophages. Proc. Natl Acad. Sci. USA 102, 5174–5179 (2005).

    CAS  Article  Google Scholar 

  30. 30.

    Westra, E. R. et al. Parasite exposure drives selective evolution of constitutive versus inducible defense. Curr. Biol. 25, 1043–1049 (2015).

    CAS  Article  Google Scholar 

  31. 31.

    Lange, S. J., Alkhnbashi, O. S., Rose, D., Will, S. & Backofen, R. CRISPRmap: an automated classification of repeat conservation in prokaryotic adaptive immune systems. Nucleic Acids Res. 41, 8034–8044 (2013).

    CAS  Article  Google Scholar 

  32. 32.

    Mann, B. A. & Slauch, J. M. Transduction of low-copy number plasmids by bacteriophage P22. Genetics 146, 447–456 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Heler, R. et al. Cas9 specifies functional viral targets during CRISPR–Cas adaptation. Nature 519, 199–202 (2015).

    CAS  Article  Google Scholar 

  34. 34.

    De Paepe, M. et al. Temperate phages acquire DNA from defective prophages by relaxed homologous recombination: the role of Rad52-like recombinases. PLoS Genet. 10, e1004181 (2014).

    Article  Google Scholar 

  35. 35.

    Lopes, A., Amarir-Bouhram, J., Faure, G., Petit, M. A. & Guerois, R. Detection of novel recombinases in bacteriophage genomes unveils Rad52, Rad51 and Gp2.5 remote homologs. Nucleic Acids Res. 38, 3952–3962 (2010).

    CAS  Article  Google Scholar 

  36. 36.

    Modell, J. W., Jiang, W. & Marraffini, L. A. CRISPR–Cas systems exploit viral DNA injection to establish and maintain adaptive immunity. Nature 544, 101–104 (2017).

    CAS  Article  Google Scholar 

  37. 37.

    Polz, M. F., Alm, E. J. & Hanage, W. P. Horizontal gene transfer and the evolution of bacterial and archaeal population structure. Trends Genet. 29, 170–175 (2013).

    CAS  Article  Google Scholar 

  38. 38.

    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).

    CAS  Article  Google Scholar 

  39. 39.

    Ho Sui, S. J., Fedynak, A., Hsiao, W. W., Langille, M. G. & Brinkman, F. S. The association of virulence factors with genomic islands. PLoS ONE 4, e8094 (2009).

    Article  Google Scholar 

  40. 40.

    van Houte, S. et al. The diversity-generating benefits of a prokaryotic adaptive immune system. Nature 532, 385–388 (2016).

    Article  Google Scholar 

  41. 41.

    Minot, S. et al. The human gut virome: inter-individual variation and dynamic response to diet. Genome Res. 21, 1616–1625 (2011).

    CAS  Article  Google Scholar 

  42. 42.

    Seed, K. D., Lazinski, D. W., Calderwood, S. B. & Camilli, A. A bacteriophage encodes its own CRISPR/Cas adaptive response to evade host innate immunity. Nature 494, 489–491 (2013).

    CAS  Article  Google Scholar 

  43. 43.

    Sebaihia, M. et al. The multidrug-resistant human pathogen Clostridium difficile has a highly mobile, mosaic genome. Nat. Genet. 38, 779–786 (2006).

    Article  Google Scholar 

  44. 44.

    Peters, J. E., Makarova, K. S., Shmakov, S. & Koonin, E. V. Recruitment of CRISPR–Cas systems by Tn7-like transposons. Proc. Natl Acad. Sci. USA 114, E7358–E7366 (2017).

    CAS  Article  Google Scholar 

  45. 45.

    Hatoum-Aslan, A., Maniv, I. & Marraffini, L. A. Mature clustered, regularly interspaced, short palindromic repeats RNA (crRNA) length is measured by a ruler mechanism anchored at the precursor processing site. Proc. Natl Acad. Sci. USA 108, 21218–21222 (2011).

    CAS  Article  Google Scholar 

  46. 46.

    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).

    CAS  Article  Google Scholar 

  47. 47.

    Kreiswirth, B. N. et al. The toxic shock syndrome exotoxin structural gene is not detectably transmitted by a prophage. Nature 305, 709–712 (1983).

    CAS  Article  Google Scholar 

  48. 48.

    Horinouchi, S. & Weisblum, B. Nucleotide sequence and functional map of pC194, a plasmid that specifies inducible chloramphenicol resistance. J. Bacteriol. 150, 815–825 (1982).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. 49.

    Schneewind, O., Model, P. & Fischetti, V. A. Sorting of protein A to the staphylococcal cell wall. Cell 70, 267–281 (1992).

    CAS  Article  Google Scholar 

  50. 50.

    Hynes, A. P. et al. Detecting natural adaptation of the Streptococcus thermophilus CRISPR–Cas systems in research and classroom settings. Nat. Protoc. 12, 547–565 (2017).

    CAS  Article  Google Scholar 

  51. 51.

    Cady, K. C., Bondy-Denomy, J., Heussler, G. E., Davidson, A. R. & O’Toole, G. A. The CRISPR/Cas adaptive immune system of Pseudomonas aeruginosa mediates resistance to naturally occurring and engineered phages. J. Bacteriol. 194, 5728–5738 (2012).

    CAS  Article  Google Scholar 

  52. 52.

    Goldberg, G. W., Jiang, W., Bikard, D. & Marraffini, L. A. Conditional tolerance of temperate phages via transcription-dependent CRISPR–Cas targeting. Nature 514, 633–637 (2014).

    CAS  Article  Google Scholar 

  53. 53.

    McGinn, J. & Marraffini, L. A. CRISPR–Cas systems optimize their immune response by specifying the site of spacer integration. Mol. Cell 64, 616–623 (2016).

    CAS  Article  Google Scholar 

  54. 54.

    Martinez-Garcia, E., Calles, B., Arevalo-Rodriguez, M. & de Lorenzo, V. pBAM1: an all-synthetic genetic tool for analysis and construction of complex bacterial phenotypes. BMC Microbiol. 11, 38 (2011).

    CAS  Article  Google Scholar 

  55. 55.

    Husmann, L. K., Scott, J. R., Lindahl, G. & Stenberg, L. Expression of the Arp protein, a member of the M protein family, is not sufficient to inhibit phagocytosis of Streptococcus pyogenes. Infect. Immun. 63, 345–348 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. 56.

    Horinouchi, S. & Weisblum, B. Nucleotide sequence and functional map of pE194, a plasmid that specifies inducible resistance to macrolide, lincosamide, and streptogramin type B antibodies. J. Bacteriol. 150, 804–814 (1982).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. 57.

    Letort, C. & Juillard, V. Development of a minimal chemically-defined medium for the exponential growth of Streptococcus thermophilus. J. Appl. Microbiol. 91, 1023–1029 (2001).

    CAS  Article  Google Scholar 

  58. 58.

    Fontaine, L. et al. Development of a versatile procedure based on natural transformation for marker-free targeted genetic modification in Streptococcus thermophilus. Appl. Environ. Microbiol. 76, 7870–7877 (2010).

    CAS  Article  Google Scholar 

  59. 59.

    Bikard, D. et al. Exploiting CRISPR–Cas nucleases to produce sequence-specific antimicrobials. Nat. Biotechnol. 32, 1146–1150 (2014).

    CAS  Article  Google Scholar 

  60. 60.

    Wang, H., Claveau, D., Vaillancourt, J. P., Roemer, T. & Meredith, T. C. High-frequency transposition for determining antibacterial mode of action. Nat. Chem. Biol. 7, 720–729 (2011).

    CAS  Article  Google Scholar 

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We thank J. W. Modell, J. T. Rostol and A. M. Pham for helpful discussion and critical reading of the manuscript. J. W. Modell (The Rockefeller University) also provided the strain pAV293-296. A.V. is supported by the Arnold O. Beckman Postdoctoral Fellowship. L.A.M. is supported by the Rita Allen Scholars Program and an NIH Director’s Pioneer Award (DP1GM128184-01). The work carried out by E.R.W. and S.M. was supported by the Biotechnology and Biological Sciences Research Council (BB/N017412/1) and Natural Environment Research Council (NE/M018350/1).

Author information




A.V. and L.A.M. conceived the study. A.V., S.M., R.B., E.R.W. and L.A.M. designed the experiments. A.V. executed the experimental work. S.M. executed the experimental work with P.aeruginosa. A.V., S.M., R.B., E.R.W. and L.A.M. wrote the paper.

Corresponding author

Correspondence to Luciano A. Marraffini.

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Competing interests

L.A.M. is a cofounder and Scientific Advisory Board member of Intellia Therapeutics and a cofounder of Eligo Biosciences. R.B. is a cofounder and Scientific Advisory Board member of Intellia Therapeutics, a cofounder of Locus Biosciences, an advisor to Inari Ag and a shareholder of DuPont and Caribou Biosciences. A.V., E.R.W. and S.M. declare no competing interests.

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Supplementary Information

Supplementary Figures 1–9, Supplementary Tables 1 and 2, legend for Supplementary Dataset and Supplementary References.

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Supplementary Data Set 1

Next-generation sequencing data used to generate Fig. 2a and Supplementary Fig. 2a–c.

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Varble, A., Meaden, S., Barrangou, R. et al. Recombination between phages and CRISPR−cas loci facilitates horizontal gene transfer in staphylococci. Nat Microbiol 4, 956–963 (2019).

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