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Self versus non-self discrimination during CRISPR RNA-directed immunity



All immune systems must distinguish self from non-self to repel invaders without inducing autoimmunity. Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci protect bacteria and archaea from invasion by phage and plasmid DNA through a genetic interference pathway1,2,3,4,5,6,7,8,9. CRISPR loci are present in 40% and 90% of sequenced bacterial and archaeal genomes, respectively10, and evolve rapidly, acquiring new spacer sequences to adapt to highly dynamic viral populations1,11,12,13. Immunity requires a sequence match between the invasive DNA and the spacers that lie between CRISPR repeats1,2,3,4,5,6,7,8,9. Each cluster is genetically linked to a subset of the cas (CRISPR-associated) genes14,15,16 that collectively encode >40 families of proteins involved in adaptation and interference. CRISPR loci encode small CRISPR RNAs (crRNAs) that contain a full spacer flanked by partial repeat sequences2,17,18,19. CrRNA spacers are thought to identify targets by direct Watson–Crick pairing with invasive ‘protospacer’ DNA2,3, but how they avoid targeting the spacer DNA within the encoding CRISPR locus itself is unknown. Here we have defined the mechanism of CRISPR self/non-self discrimination. In Staphylococcus epidermidis, target/crRNA mismatches at specific positions outside of the spacer sequence license foreign DNA for interference, whereas extended pairing between crRNA and CRISPR DNA repeats prevents autoimmunity. Hence, this CRISPR system uses the base-pairing potential of crRNAs not only to specify a target, but also to spare the bacterial chromosome from interference. Differential complementarity outside of the spacer sequence is a built-in feature of all CRISPR systems, indicating that this mechanism is a broadly applicable solution to the self/non-self dilemma that confronts all immune pathways.

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Figure 1: Protection of nes target by spacer flanking sequences.
Figure 2: Complementarity between crRNA and target DNA flanking sequences is required for protection.
Figure 3: Mutations in upstream flanking sequences of CRISPR spacers elicit autoimmunity.
Figure 4: Requirements for targeting and protection during CRISPR immunity.


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

    Article  ADS  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  4. Sorek, R., Kunin, V. & Hugenholtz, P. CRISPR–a widespread system that provides acquired resistance against phages in bacteria and archaea. Nature Rev. Microbiol. 6, 181–186 (2008)

    Article  CAS  Google Scholar 

  5. Bolotin, A., Quinquis, B., Sorokin, A. & Ehrlich, S. D. Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology 151, 2551–2561 (2005)

    Article  CAS  Google Scholar 

  6. Pourcel, C., Salvignol, G. & Vergnaud, G. CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies. Microbiology 151, 653–663 (2005)

    Article  CAS  Google Scholar 

  7. Lillestøl, R. K., Redder, P., Garrett, R. A. & Brugger, K. A putative viral defence mechanism in archaeal cells. Archaea 2, 59–72 (2006)

    Article  Google Scholar 

  8. Mojica, F. J., Diez-Villasenor, C., Garcia-Martinez, J. & Soria, E. Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J. Mol. Evol. 60, 174–182 (2005)

    Article  ADS  CAS  Google Scholar 

  9. van der Oost, J., Jore, M. M., Westra, E. R., Lundgren, M. & Brouns, S. J. CRISPR-based adaptive and heritable immunity in prokaryotes. Trends Biochem. Sci. 34, 401–407 (2009)

    Article  CAS  Google Scholar 

  10. Grissa, I., Vergnaud, G. & Pourcel, C. The CRISPRdb database and tools to display CRISPRs and to generate dictionaries of spacers and repeats. BMC Bioinformatics 8, 172 (2007)

    Article  Google Scholar 

  11. Andersson, A. F. & Banfield, J. F. Virus population dynamics and acquired virus resistance in natural microbial communities. Science 320, 1047–1050 (2008)

    Article  ADS  CAS  Google Scholar 

  12. Deveau, H. et al. Phage response to CRISPR-encoded resistance in Streptococcus thermophilus . J. Bacteriol. 190, 1390–1400 (2008)

    Article  CAS  Google Scholar 

  13. van der Ploeg, J. R. Analysis of CRISPR in Streptococcus mutans suggests frequent occurrence of acquired immunity against infection by M102-like bacteriophages. Microbiology 155, 1966–1976 (2009)

    Article  CAS  Google Scholar 

  14. 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)

    Article  CAS  Google Scholar 

  15. 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  ADS  Google Scholar 

  16. Makarova, K. S., Grishin, N. V., Shabalina, S. A., Wolf, Y. I. & Koonin, E. V. A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action. Biol. Direct 1, 7 (2006)

    Article  Google Scholar 

  17. Hale, C., Kleppe, K., Terns, R. M. & Terns, M. P. Prokaryotic silencing (psi)RNAs in Pyrococcus furiosus . RNA 14, 2572–2579 (2008)

    Article  CAS  Google Scholar 

  18. Tang, T. H. et al. Identification of 86 candidates for small non-messenger RNAs from the archaeon Archaeoglobus fulgidus . Proc. Natl Acad. Sci. USA 99, 7536–7541 (2002)

    Article  ADS  CAS  Google Scholar 

  19. Tang, T. H. et al. Identification of novel non-coding RNAs as potential antisense regulators in the archaeon Sulfolobus solfataricus . Mol. Microbiol. 55, 469–481 (2005)

    Article  CAS  Google Scholar 

  20. Gill, S. R. et al. Insights on evolution of virulence and resistance from the complete genome analysis of an early methicillin-resistant Staphylococcus aureus strain and a biofilm-producing methicillin-resistant Staphylococcus epidermidis strain. J. Bacteriol. 187, 2426–2438 (2005)

    Article  CAS  Google Scholar 

  21. Climo, M. W., Sharma, V. K. & Archer, G. L. Identification and characterization of the origin of conjugative transfer (oriT) and a gene (nes) encoding a single-stranded endonuclease on the staphylococcal plasmid pGO1. J. Bacteriol. 178, 4975–4983 (1996)

    Article  CAS  Google Scholar 

  22. Diep, B. A. et al. Complete genome sequence of USA300, an epidemic clone of community-acquired meticillin-resistant Staphylococcus aureus . Lancet 367, 731–739 (2006)

    Article  CAS  Google Scholar 

  23. Weigel, L. M. et al. Genetic analysis of a high-level vancomycin-resistant isolate of Staphylococcus aureus . Science 302, 1569–1571 (2003)

    Article  ADS  CAS  Google Scholar 

  24. Lillestøl, R. K. et al. CRISPR families of the crenarchaeal genus Sulfolobus: bidirectional transcription and dynamic properties. Mol. Microbiol. 72, 259–272 (2009)

    Article  Google Scholar 

  25. Semenova, E., Nagornykh, M., Pyatnitskiy, M., Artamonova, I. I. & Severinov, K. Analysis of CRISPR system function in plant pathogen Xanthomonas oryzae . FEMS Microbiol. Lett. 296, 110–116 (2009)

    Article  CAS  Google Scholar 

  26. Shah, S. A., Hansen, N. R. & Garrett, R. A. Distribution of CRISPR spacer matches in viruses and plasmids of crenarchaeal acidothermophiles and implications for their inhibitory mechanism. Biochem. Soc. Trans. 37, 23–28 (2009)

    Article  CAS  Google Scholar 

  27. Mojica, F. J., Diez-Villasenor, C., Garcia-Martinez, J. & Almendros, C. Short motif sequences determine the targets of the prokaryotic CRISPR defence system. Microbiology 155, 733–740 (2009)

    Article  CAS  Google Scholar 

  28. Sugimoto, N., Nakano, M. & Nakano, S. Thermodynamics-structure relationship of single mismatches in RNA/DNA duplexes. Biochemistry 39, 11270–11281 (2000)

    Article  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  32. Bae, T. & Schneewind, O. Allelic replacement in Staphylococcus aureus with inducible counter-selection. Plasmid 55, 58–63 (2006)

    Article  CAS  Google Scholar 

  33. Morton, T. M., Johnston, J. L., Patterson, J. & Archer, G. L. Characterization of a conjugative staphylococcal mupirocin resistance plasmid. Antimicrob. Agents Chemother. 39, 1272–1280 (1995)

    Article  CAS  Google Scholar 

  34. Pall, G. S., Codony-Servat, C., Byrne, J., Ritchie, L. & Hamilton, A. Carbodiimide-mediated cross-linking of RNA to nylon membranes improves the detection of siRNA, miRNA and piRNA by northern blot. Nucleic Acids Res. 35, e60 (2007)

    Article  Google Scholar 

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We thank N. Fang for cloning assistance and members of our laboratory for critical reading of the manuscript. We thank V. Gerbasi and J. Marques for experimental advice. L.A.M. is a Fellow of The Jane Coffin Childs Memorial Fund for Medical Research. This work was supported by a grant from the National Institutes of Health, USA, to E.J.S.

Author Contributions L.A.M. designed experiments with input from E.J.S.; L.A.M. conducted experiments. L.A.M. and E.J.S. analysed data, interpreted experiments and wrote the paper.

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Correspondence to Luciano A. Marraffini or Erik J. Sontheimer.

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Marraffini, L., Sontheimer, E. Self versus non-self discrimination during CRISPR RNA-directed immunity. Nature 463, 568–571 (2010).

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