RNA-guided genetic silencing systems in bacteria and archaea

Abstract

Clustered regularly interspaced short palindromic repeat (CRISPR) are essential components of nucleic-acid-based adaptive immune systems that are widespread in bacteria and archaea. Similar to RNA interference (RNAi) pathways in eukaryotes, CRISPR-mediated immune systems rely on small RNAs for sequence-specific detection and silencing of foreign nucleic acids, including viruses and plasmids. However, the mechanism of RNA-based bacterial immunity is distinct from RNAi. Understanding how small RNAs are used to find and destroy foreign nucleic acids will provide new insights into the diverse mechanisms of RNA-controlled genetic silencing systems.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Parallels and distinctions between CRISPR RNA-guided silencing systems and RNAi.
Figure 2: Diversity of CRISPR-mediated adaptive immune systems in bacteria and archaea.
Figure 3: Steps leading to new spacer integration.
Figure 4: Diverse mechanisms of CRISPR RNA biogenesis.

References

  1. 1

    Hoskisson, P. A. & Smith, M. C. Hypervariation and phase variation in the bacteriophage 'resistome'. Curr. Opin. Microbiol. 10, 396–400 (2007).

    CAS  Article  Google Scholar 

  2. 2

    Rodriguez-Valera, F. et al. Explaining microbial population genomics through phage predation. Nature Rev. Microbiol. 7, 828–836 (2009).

    CAS  Article  Google Scholar 

  3. 3

    Weinbauer, M. G. Ecology of prokaryotic viruses. FEMS Microbiol. Rev. 28, 127–181 (2004).

    CAS  Article  Google Scholar 

  4. 4

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

    CAS  Article  Google Scholar 

  5. 5

    Stern, A. & Sorek, R. The phage-host arms race: shaping the evolution of microbes. Bioessays 33, 43–51 (2010).

    Article  Google Scholar 

  6. 6

    Al-Attar, S., Westra, E. R., van der Oost, J. & Brouns, S. J. Clustered regularly interspaced short palindromic repeats (CRISPRs): the hallmark of an ingenious antiviral defense mechanism in prokaryotes. Biol. Chem. 392, 277–289 (2011).

    CAS  Article  Google Scholar 

  7. 7

    Deveau, H., Garneau, J. E. & Moineau, S. CRISPR/Cas system and its role in phage-bacteria interactions. Annu. Rev. Microbiol. 64, 475–493 (2010).

    CAS  Article  Google Scholar 

  8. 8

    Horvath, P. & Barrangou, R. CRISPR/Cas, the immune system of bacteria and archaea. Science 327, 167–170 (2010).

    ADS  CAS  Article  Google Scholar 

  9. 9

    Karginov, F. V. & Hannon, G. J. The CRISPR system: small RNA-guided defense in bacteria and archaea. Mol. Cell 37, 7–19 (2010).

    CAS  Article  Google Scholar 

  10. 10

    Makarova, K. S. et al. Evolution and classification of the CRISPR-Cas systems. Nature Rev. Microbiol. 9, 467–477 (2011).

    CAS  Article  Google Scholar 

  11. 11

    Marraffini, L. A. & Sontheimer, E. J. CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea. Nature Rev. Genet. 11, 181–190 (2010).

    CAS  Article  Google Scholar 

  12. 12

    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).

    CAS  Article  Google Scholar 

  13. 13

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

    ADS  CAS  Article  Google Scholar 

  14. 14

    Barrangou, R. et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science 315, 1709–1712 (2007). This study provided the first direct evidence for CRISPR immune system function, demonstrating that new spacer acquisition provides resistance against future phage infection in a cas gene-dependent manner.

    ADS  CAS  Article  Google Scholar 

  15. 15

    Garneau, J. E. et al. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature 468, 67–71 (2010). This study showed that CRISPRs can acquire new spacers upon plasmid challenge and provided the first example of crRNA-guided cleavage of double-stranded DNA in vivo.

    ADS  CAS  Article  Google Scholar 

  16. 16

    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 

  17. 17

    Deltcheva, E. et al. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 471, 602–607 (2011). This study identified a new CRISPR RNA processing pathway that involves a trans -acting RNA complementary to the CRISPR repeat sequence and demonstrated that processing of this duplex is mediated by cellular RNase III.

    ADS  CAS  Article  Google Scholar 

  18. 18

    Gesner, E. M., Schellenberg, M. J., Garside, E. L., George, M. M. & MacMillan, A. M. Recognition and maturation of effector RNAs in a CRISPR interference pathway Nature Struct. Mol. Biol. 18, 688–692 (2011).

    CAS  Article  Google Scholar 

  19. 19

    Haurwitz, R. E., Jinek, M., Wiedenheft, B., Zhou, K. & Doudna, J. A. Sequence- and structure-specific RNA processing by a CRISPR endonuclease. Science 329, 1355–1358 (2010).

    ADS  CAS  Article  Google Scholar 

  20. 20

    Sashital, D. G., Jinek, M. & Doudna, J. A. An RNA induced conformational change required for CRISPR RNA cleavage by the endonuclease Cse3. Nature Struct. Mol. Biol. 18, 680–687 (2011).

    CAS  Article  Google Scholar 

  21. 21

    Wang, R., Preamplume, G., Terns, M. P., Terns, R. M. & Li, H. Interaction of the Cas6 riboendonuclease with CRISPR RNAs: recognition and cleavage. Structure 19, 257–264 (2011).

    CAS  Article  Google Scholar 

  22. 22

    Brouns, S. J. et al. Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 321, 960–964 (2008). This paper revealed that long CRISPR RNA transcripts are processed into small RNAs (crRNAs) by a dedicated endoribonuclease and that the processed RNAs are bound by a Cas protein complex (Cascade), which together with Cas3 are required for phage protection.

    ADS  CAS  Article  Google Scholar 

  23. 23

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

    CAS  Article  Google Scholar 

  24. 24

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

    CAS  Article  Google Scholar 

  25. 25

    Lintner, N. G. et al. Structural and functional characterization of an archaeal clustered regularly interspaced short palindromic repeat (crispr)-associated complex for antiviral defense (CASCADE). J. Biol. Chem. 286, 21643–21656 (2011).

    CAS  Article  Google Scholar 

  26. 26

    Wiedenheft, B. et al. Structures of the RNA-guided surveillance complex from a bacterial immune system. Nature 477, 486–489 (2011). This paper presented the first sub-nanometer resolution structures of Cascade, showing how the crRNA is accommodated within a large ribonucleoprotein complex that is involved in foreign nucleic acid surveillance.

    ADS  CAS  Article  Google Scholar 

  27. 27

    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).

    ADS  CAS  Article  Google Scholar 

  28. 28

    Ishino, Y., Shinagawa, H., Makino, K., Amemura, M. & Nakata, A. Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product. J. Bacteriol. 169, 5429–5433 (1987).

    CAS  Article  Google Scholar 

  29. 29

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

    CAS  Article  Google Scholar 

  30. 30

    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).

    ADS  CAS  Article  Google Scholar 

  31. 31

    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).

    CAS  Article  Google Scholar 

  32. 32

    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 

  33. 33

    Grissa, I., Vergnaud, G. & Pourcel, C. CRISPRFinder: a web tool to identify clustered regularly interspaced short palindromic repeats. Nucleic Acids Res. 35, W52–W57 (2007).

    Article  Google Scholar 

  34. 34

    Rousseau, C., Gonnet, M., Le Romancer, M. & Nicolas, J. CRISPI: a CRISPR interactive database. Bioinformatics 25, 3317–3318 (2009).

    CAS  Article  Google Scholar 

  35. 35

    Kunin, V., Sorek, R. & Hugenholtz, P. Evolutionary conservation of sequence and secondary structures in CRISPR repeats. Genome Biol. 8, R61 (2007).

    Article  Google Scholar 

  36. 36

    Bland, C. et al. CRISPR recognition tool (CRT): a tool for automatic detection of clustered regularly interspaced palindromic repeats. BMC Bioinformatics 8, 209 (2007).

    Article  Google Scholar 

  37. 37

    Dsouza, M., Larsen, N. & Overbeek, R. Searching for patterns in genomic data. Trends Genet. 13, 497–498 (1997).

    CAS  Article  Google Scholar 

  38. 38

    Edgar, R. C. PILER-CR: fast and accurate identification of CRISPR repeats. BMC Bioinformatics 8, 18 (2007).

    Article  Google Scholar 

  39. 39

    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 

  40. 40

    Pougach, K. et al. Transcription, processing and function of CRISPR cassettes in Escherichia coli. Mol. Microbiol. 77, 1367–1379 (2010).

    CAS  Article  Google Scholar 

  41. 41

    Pul, U. et al. Identification and characterization of E. coli CRISPR-cas promoters and their silencing by H-NS. Mol. Microbiol. 75, 1495–1512 (2010).

    CAS  Article  Google Scholar 

  42. 42

    Westra, E. R. et al. H-NS-mediated repression of CRISPR-based immunity in Escherichia coli K12 can be relieved by the transcription activator LeuO. Mol. Microbiol. 77, 1380–1393 (2010).

    CAS  Article  Google Scholar 

  43. 43

    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).

    ADS  Article  Google Scholar 

  44. 44

    Sinkunas, T. et al. Cas3 is a single-stranded DNA nuclease and ATP-dependent helicase in the CRISPR/Cas immune system. EMBO J. 30, 1335–1342 (2011).

    CAS  Article  Google Scholar 

  45. 45

    Han, D. & Krauss, G. Characterization of the endonuclease SSO2001 from Sulfolobus solfataricus P2. FEBS Lett. 583, 771–776 (2009).

    CAS  Article  Google Scholar 

  46. 46

    Mulepati, S. & Bailey, S. Structural and biochemical analysis of the nuclease domain of the clustered regularly interspaced short palindromic repeat (CRISPR) associated protein 3(CAS3). J. Biol. Chem. 286, 31896–31903 (2011).

    CAS  Article  Google Scholar 

  47. 47

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

    ADS  CAS  Article  Google Scholar 

  48. 48

    Hale, C. R. et al. RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex. Cell 139, 945–956 (2009). Most CRISPR systems appear to target DNA, but this article reported the isolation of a multisubunit ribonucleoprotein complex (Cmr-complex) from P. furriosus that cleaves target RNAs (not DNA) at a fixed distance from the 3′ end of the crRNA-guide.

    CAS  Article  Google Scholar 

  49. 49

    Tyson, G. W. & Banfield, J. F. Rapidly evolving CRISPRs implicated in acquired resistance of microorganisms to viruses. Environ. Microbiol. 10, 200–207 (2008).

    CAS  Google Scholar 

  50. 50

    Snyder, J. C., Bateson, M. M., Lavin, M. & Young, M. J. Use of cellular CRISPR (clusters of regularly interspaced short palindromic repeats) spacer-based microarrays for detection of viruses in environmental samples. Appl Environ Microbiol 76, 7251–7258 (2010).

    CAS  Google Scholar 

  51. 51

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

    CAS  Article  Google Scholar 

  52. 52

    Horvath, P. et al. Diversity, activity, and evolution of CRISPR loci in Streptococcus thermophilus. J. Bacteriol. 190, 1401–1412 (2008).

    CAS  Article  Google Scholar 

  53. 53

    Sapranauskas, R. et al. The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli. Nucleic Acids Res. 39, 9275–9282 (2011).

    CAS  Article  Google Scholar 

  54. 54

    Babu, M. et al. A dual function of the CRISPR-Cas system in bacterial antivirus immunity and DNA repair. Mol. Microbiol. 79, 484–502 (2011).

    CAS  Article  Google Scholar 

  55. 55

    Han, D., Lehmann, K. & Krauss, G. SSO1450--a CAS1 protein from Sulfolobus solfataricus P2 with high affinity for RNA and DNA. FEBS Lett. 583, 1928–1932 (2009).

    CAS  Article  Google Scholar 

  56. 56

    Wiedenheft, B. et al. Structural basis for DNase activity of a conserved protein implicated in CRISPR-mediated antiviral defense. Structure 17, 904–912 (2009).

    CAS  Article  Google Scholar 

  57. 57

    Chen, C. S. et al. A proteome chip approach reveals new DNA damage recognition activities in Escherichia coli. Nature Methods 5, 69–74 (2008).

    CAS  Article  Google Scholar 

  58. 58

    Aguilera, A. The connection between transcription and genomic instability. EMBO J. 21, 195–201 (2002).

    CAS  Article  Google Scholar 

  59. 59

    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).

    CAS  Article  Google Scholar 

  60. 60

    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).

    ADS  CAS  Article  Google Scholar 

  61. 61

    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).

    ADS  CAS  Article  Google Scholar 

  62. 62

    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).

    CAS  Article  Google Scholar 

  63. 63

    Ebihara, A. et al. Crystal structure of hypothetical protein TTHB192 from Thermus thermophilus HB8 reveals a new protein family with an RNA recognition motif-like domain. Protein Sci. 15, 1494–1499 (2006).

    CAS  Article  Google Scholar 

  64. 64

    Carte, J., Pfister, N. T., Compton, M. M., Terns, R. M. & Terns, M. P. Binding and cleavage of CRISPR RNA by Cas6. RNA 16, 2181–2188 (2010).

    CAS  Article  Google Scholar 

  65. 65

    Gudbergsdottir, S. et al. Dynamic properties of the Sulfolobus CRISPR/Cas and CRISPR/Cmr systems when challenged with vector-borne viral and plasmid genes and protospacers. Mol. Microbiol. 79, 35–49 (2011)

    CAS  Article  Google Scholar 

  66. 66

    Manica, A., Zebec, Z., Teichmann, D. & Schleper, C. In vivo activity of CRISPR-mediated virus defence in a hyperthermophilic archaeon. Mol. Microbiol. 80, 481–491 (2011).

    CAS  Article  Google Scholar 

  67. 67

    Marraffini, L. A. & Sontheimer, E. J. Self versus non-self discrimination during CRISPR RNA-directed immunity. Nature 463, 568–571 (2010). This study identified a mechanism for distingishing self from non-self, which relies on base-pairing potential in regions outside the crRNA spacer sequence.

    ADS  CAS  Article  Google Scholar 

  68. 68

    Bartel, D. P. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281–297 (2004).

    CAS  Article  Google Scholar 

  69. 69

    Parker, J. S., Parizotto, E. A., Wang, M., Roe, S. M. & Barford, D. Enhancement of the seed-target recognition step in RNA silencing by a PIWI/MID domain protein. Mol. Cell 33, 204–214 (2009).

    CAS  Article  Google Scholar 

  70. 70

    Beloglazova, N. et al. Structure and activity of the Cas3 HD nuclease MJ0384, an effector enzyme of the CRISPR interference. EMBO J. 30, 4616–4627 (2011).

    CAS  Article  Google Scholar 

  71. 71

    Groenen, P. M., Bunschoten, A. E., van Soolingen, D. & van Embden, J. D. Nature of DNA polymorphism in the direct repeat cluster of Mycobacterium tuberculosis; application for strain differentiation by a novel typing method. Mol. Microbiol. 10, 1057–1065 (1993).

    CAS  Article  Google Scholar 

  72. 72

    Mokrousov, I., Limeschenko, E., Vyazovaya, A. & Narvskaya, O. Corynebacterium diphtheriae spoligotyping based on combined use of two CRISPR loci. Biotechnol. J. 2, 901–906 (2007).

    CAS  Article  Google Scholar 

  73. 73

    Liu, F. et al. Novel virulence gene and clustered regularly interspaced short palindromic repeat (CRISPR) multilocus sequence typing scheme for subtyping of the major serovars of Salmonella enterica subsp. enterica. Appl. Environ. Microbiol. 77, 1946–1956 (2011).

    CAS  Article  Google Scholar 

  74. 74

    Cady, K. C. & O'Toole, G. A. Non-identity targeting of yersinia-subtype CRISPR-prophage interaction requires the Csy and Cas3 proteins. J. Bacteriol. 193, 3433–3445 (2011).

    CAS  Article  Google Scholar 

  75. 75

    Zegans, M. E. et al. Interaction between bacteriophage DMS3 and host CRISPR region inhibits group behaviors of Pseudomonas aeruginosa. J. Bacteriol. 191, 210–219 (2009).

    CAS  Article  Google Scholar 

  76. 76

    Obbard, D. J., Gordon, K. H. J., Buck, A. H. & Jiggins, F. M. The evolution of RNAi as a defence against viruses and transposable elements. Philos. Trans. R. Soc. B Biol. Sci. 364, 99–115 (2009).

    CAS  Article  Google Scholar 

  77. 77

    Aravin, A. A., Hannon, G. J. & Brennecke, J. The Piwi-piRNA pathway provides an adaptive defense in the transposon arms race. Science 318, 761–764 (2007).

    ADS  CAS  Article  Google Scholar 

  78. 78

    Aravin, A. A. et al. Double-stranded RNA-mediated silencing of genomic tandem repeats and transposable elements in the D-melanogaster germline. Curr Biol 11, 1017–1027 (2001).

    CAS  Article  Google Scholar 

  79. 79

    Bartel, D. P. MicroRNAs: target recognition and regulatory functions. Cell 136, 215–233 (2009).

    CAS  Article  Google Scholar 

  80. 80

    Guo, H., Ingolia, N. T., Weissman, J. S. & Bartel, D. P. Mammalian microRNAs predominantly act to decrease target mRNA levels. Nature 466, 835–840 (2010).

    ADS  CAS  Article  Google Scholar 

Download references

Acknowledgements

B.W. is a Howard Hughes Medical Institute Fellow of the Life Sciences Research Foundation. S.H.S. acknowledges support from the National Science Foundation and National Defense Science & Engineering Graduate Research Fellowship programs. J.A.D. is an Investigator of the Howard Hughes Medical Institute.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Jennifer A. Doudna.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Additional information

Reprints and permissions information is available at www.nature.com/reprints

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Wiedenheft, B., Sternberg, S. & Doudna, J. RNA-guided genetic silencing systems in bacteria and archaea. Nature 482, 331–338 (2012). https://doi.org/10.1038/nature10886

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.