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.
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Hoskisson, P. A. & Smith, M. C. Hypervariation and phase variation in the bacteriophage 'resistome'. Curr. Opin. Microbiol. 10, 396–400 (2007).
Rodriguez-Valera, F. et al. Explaining microbial population genomics through phage predation. Nature Rev. Microbiol. 7, 828–836 (2009).
Weinbauer, M. G. Ecology of prokaryotic viruses. FEMS Microbiol. Rev. 28, 127–181 (2004).
Labrie, S. J., Samson, J. E. & Moineau, S. Bacteriophage resistance mechanisms. Nature Rev. Microbiol. 8, 317–327 (2010).
Stern, A. & Sorek, R. The phage-host arms race: shaping the evolution of microbes. Bioessays 33, 43–51 (2010).
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).
Deveau, H., Garneau, J. E. & Moineau, S. CRISPR/Cas system and its role in phage-bacteria interactions. Annu. Rev. Microbiol. 64, 475–493 (2010).
Horvath, P. & Barrangou, R. CRISPR/Cas, the immune system of bacteria and archaea. Science 327, 167–170 (2010).
Karginov, F. V. & Hannon, G. J. The CRISPR system: small RNA-guided defense in bacteria and archaea. Mol. Cell 37, 7–19 (2010).
Makarova, K. S. et al. Evolution and classification of the CRISPR-Cas systems. Nature Rev. Microbiol. 9, 467–477 (2011).
Marraffini, L. A. & Sontheimer, E. J. CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea. Nature Rev. Genet. 11, 181–190 (2010).
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).
Andersson, A. F. & Banfield, J. F. Virus population dynamics and acquired virus resistance in natural microbial communities. Science 320, 1047–1050 (2008).
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.
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.
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).
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.
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).
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).
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).
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).
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.
Hale, C., Kleppe, K., Terns, R. M. & Terns, M. P. Prokaryotic silencing (psi)RNAs in Pyrococcus furiosus. RNA 14, 2572–2579 (2008).
Jore, M. M. et al. Structural basis for CRISPR RNA-guided DNA recognition by Cascade. Nature Struct. Mol. Biol. 18, 529–536 (2011).
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).
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.
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).
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).
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).
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).
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).
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).
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).
Rousseau, C., Gonnet, M., Le Romancer, M. & Nicolas, J. CRISPI: a CRISPR interactive database. Bioinformatics 25, 3317–3318 (2009).
Kunin, V., Sorek, R. & Hugenholtz, P. Evolutionary conservation of sequence and secondary structures in CRISPR repeats. Genome Biol. 8, R61 (2007).
Bland, C. et al. CRISPR recognition tool (CRT): a tool for automatic detection of clustered regularly interspaced palindromic repeats. BMC Bioinformatics 8, 209 (2007).
Dsouza, M., Larsen, N. & Overbeek, R. Searching for patterns in genomic data. Trends Genet. 13, 497–498 (1997).
Edgar, R. C. PILER-CR: fast and accurate identification of CRISPR repeats. BMC Bioinformatics 8, 18 (2007).
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).
Pougach, K. et al. Transcription, processing and function of CRISPR cassettes in Escherichia coli. Mol. Microbiol. 77, 1367–1379 (2010).
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).
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).
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).
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).
Han, D. & Krauss, G. Characterization of the endonuclease SSO2001 from Sulfolobus solfataricus P2. FEBS Lett. 583, 771–776 (2009).
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).
Marraffini, L. A. & Sontheimer, E. J. CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA. Science 322, 1843–1845 (2008).
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.
Tyson, G. W. & Banfield, J. F. Rapidly evolving CRISPRs implicated in acquired resistance of microorganisms to viruses. Environ. Microbiol. 10, 200–207 (2008).
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).
Deveau, H. et al. Phage response to CRISPR-encoded resistance in Streptococcus thermophilus. J. Bacteriol. 190, 1390–1400 (2008).
Horvath, P. et al. Diversity, activity, and evolution of CRISPR loci in Streptococcus thermophilus. J. Bacteriol. 190, 1401–1412 (2008).
Sapranauskas, R. et al. The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli. Nucleic Acids Res. 39, 9275–9282 (2011).
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).
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).
Wiedenheft, B. et al. Structural basis for DNase activity of a conserved protein implicated in CRISPR-mediated antiviral defense. Structure 17, 904–912 (2009).
Chen, C. S. et al. A proteome chip approach reveals new DNA damage recognition activities in Escherichia coli. Nature Methods 5, 69–74 (2008).
Aguilera, A. The connection between transcription and genomic instability. EMBO J. 21, 195–201 (2002).
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).
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).
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).
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).
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).
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).
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)
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).
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.
Bartel, D. P. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281–297 (2004).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
Bartel, D. P. MicroRNAs: target recognition and regulatory functions. Cell 136, 215–233 (2009).
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).
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.
The authors declare no competing financial interests.
Reprints and permissions information is available at www.nature.com/reprints
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
Molecular Therapy - Nucleic Acids (2020)
Diphtheria toxin‐mediated transposon‐driven poly (A)‐trapping efficiently disrupts transcriptionally silent genes in embryonic stem cells
Advanced Drug Delivery Reviews (2020)
BMC Bioinformatics (2020)