Letter | Published:

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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

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.

Additional information

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


  1. 1.

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

  2. 2.

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

  3. 3.

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

  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).

  5. 5.

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

  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).

  7. 7.

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

  8. 8.

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

  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).

  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).

  11. 11.

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

  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).

  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).

  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).

  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).

  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).

  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).

  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).

  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).

  20. 20.

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

  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).

  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).

  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).

  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).

  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).

  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).

  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).

  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).

  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).

  30. 30.

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

  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).

  32. 32.

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

  33. 33.

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

  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).

  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).

  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).

  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).

  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).

  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).

  40. 40.

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

  41. 41.

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

  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).

  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).

  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).

  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).

  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).

  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).

  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).

  49. 49.

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

  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).

  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).

  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).

  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).

  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).

  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).

  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).

  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).

  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).

  59. 59.

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

  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).

Download references


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.

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.

Correspondence to Luciano A. Marraffini.

Supplementary information

Supplementary Information

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

Reporting Summary

Supplementary Data Set 1

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

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Further reading

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.