Abstract
Since the first demonstration that CRISPR–Cas systems provide bacteria and archaea with adaptive immunity against phages and plasmids, numerous studies have yielded key insights into the molecular mechanisms governing how these systems attack and degrade foreign DNA. However, the molecular mechanisms underlying the adaptation stage, in which new immunological memory is formed, have until recently represented a major unresolved question. In this Progress article, we discuss recent discoveries that have shown both how foreign DNA is identified by the CRISPR–Cas adaptation machinery and the molecular basis for its integration into the chromosome to form an immunological memory. Furthermore, we describe the roles of each of the specific CRISPR–Cas components that are involved in memory formation, and consider current models for their evolutionary origin.
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References
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).
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).
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).
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).
Barrangou, R. et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science 315, 1709–1712 (2007).
Makarova, K. S., Aravind, L., Wolf, Y. I. & Koonin, E. V. Unification of Cas protein families and a simple scenario for the origin and evolution of CRISPR-Cas systems. Biol. Direct 6, 38 (2011).
Pougach, K. et al. Transcription, processing and function of CRISPR cassettes in Escherichia coli. Mol. Microbiol. 77, 1367–1379 (2010).
Wei, Y., Chesne, M. T., Terns, R. M. & Terns, M. P. Sequences spanning the leader-repeat junction mediate CRISPR adaptation to phage in Streptococcus thermophilus. Nucleic Acids Res. 43, 1749–1758 (2015).
Yosef, I., Goren, M. G. & Qimron, U. Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli. Nucleic Acids Res. 40, 5569–5576 (2012).
Makarova, K. S. et al. An updated evolutionary classification of CRISPR-Cas systems. Nat. Rev. Microbiol. 13, 722–736 (2015).
Brouns, S. J. et al. Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 321, 960–964 (2008).
Rouillon, C. et al. Structure of the CRISPR interference complex CSM reveals key similarities with cascade. Mol. Cell 52, 124–134 (2013).
Spilman, M. et al. Structure of an RNA silencing complex of the CRISPR-Cas immune system. Mol. Cell 52, 146–152 (2013).
Shmakov, S. et al. Discovery and functional characterization of diverse class 2 CRISPR-Cas systems. Mol. Cell 60, 385–397 (2015).
Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).
Sapranauskas, R. et al. The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli. Nucleic Acids Res. 39, 9275–9282 (2011).
Zetsche, B. et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163, 759–771 (2015).
Deltcheva, E. et al. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 471, 602–607 (2011).
Jiang, F. & Doudna, J. A. The structural biology of CRISPR-Cas systems. Curr. Opin. Struct. Biol. 30, 100–111 (2015).
Plagens, A., Richter, H., Charpentier, E. & Randau, L. DNA and RNA interference mechanisms by CRISPR-Cas surveillance complexes. FEMS Microbiol. Rev. 39, 442–463 (2015).
Sorek, R., Lawrence, C. M. & Wiedenheft, B. CRISPR-mediated adaptive immune systems in bacteria and archaea. Annu. Rev. Biochem. 82, 237–266 (2013).
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).
Arslan, Z., Hermanns, V., Wurm, R., Wagner, R. & Pul, U. Detection and characterization of spacer integration intermediates in type I-E CRISPR–Cas system. Nucleic Acids Res. 42, 7884–7893 (2014).
Datsenko, K. A. et al. Molecular memory of prior infections activates the CRISPR/Cas adaptive bacterial immunity system. Nat. Commun. 3, 945 (2012).
Diez-Villasenor, C., Guzman, N. M., Almendros, C., Garcia-Martinez, J. & Mojica, F. J. CRISPR-spacer integration reporter plasmids reveal distinct genuine acquisition specificities among CRISPR-Cas I-E variants of Escherichia coli. RNA Biol. 10, 792–802 (2013).
Fineran, P. C. & Charpentier, E. Memory of viral infections by CRISPR-Cas adaptive immune systems: acquisition of new information. Virology 434, 202–209 (2012).
Nunez, J. K. et al. Cas1–Cas2 complex formation mediates spacer acquisition during CRISPR–Cas adaptive immunity. Nat. Struct. Mol. Biol. 21, 528–534 (2014).
Nunez, J. K., Lee, A. S., Engelman, A. & Doudna, J. A. Integrase-mediated spacer acquisition during CRISPR–Cas adaptive immunity. Nature 519, 193–198 (2015).
Savitskaya, E., Semenova, E., Dedkov, V., Metlitskaya, A. & Severinov, K. High-throughput analysis of type I-E CRISPR/Cas spacer acquisition in E. coli. RNA Biol. 10, 716–725 (2013).
Shmakov, S. et al. Pervasive generation of oppositely oriented spacers during CRISPR adaptation. Nucleic Acids Res. 42, 5907–5916 (2014).
Yosef, I. et al. DNA motifs determining the efficiency of adaptation into the Escherichia coli CRISPR array. Proc. Natl Acad. Sci. USA 110, 14396–14401 (2013).
Rollie, C., Schneider, S., Brinkmann, A. S., Bolt, E. L. & White, M. F. Intrinsic sequence specificity of the Cas1 integrase directs new spacer acquisition. eLife 4, e08716 (2015).
Wei, Y., Terns, R. M. & Terns, M. P. Cas9 function and host genome sampling in type II-A CRISPR–Cas adaptation. Genes Dev. 29, 356–361 (2015).
Heler, R. et al. Cas9 specifies functional viral targets during CRISPR–Cas adaptation. Nature 519, 199–202 (2015).
Lopez-Sanchez, M. J. et al. The highly dynamic CRISPR1 system of Streptococcus agalactiae controls the diversity of its mobilome. Mol. Microbiol. 85, 1057–1071 (2012).
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).
Li, M., Wang, R., Zhao, D. & Xiang, H. Adaptation of the Haloarcula hispanica CRISPR-Cas system to a purified virus strictly requires a priming process. Nucleic Acids Res. 42, 2483–2492 (2014).
Erdmann, S. & Garrett, R. A. Selective and hyperactive uptake of foreign DNA by adaptive immune systems of an archaeon via two distinct mechanisms. Mol. Microbiol. 85, 1044–1056 (2012).
Erdmann, S., Le Moine Bauer, S. & Garrett, R. A. Inter-viral conflicts that exploit host CRISPR immune systems of Sulfolobus. Mol. Microbiol. 91, 900–917 (2014).
Hooton, S. P. & Connerton, I. F. Campylobacter jejuni acquire new host-derived CRISPR spacers when in association with bacteriophages harboring a CRISPR-like Cas4 protein. Front. Microbiol. 5, 744 (2014).
Barrangou, R. & van der Oost, J. CRISPR-Cas Systems (eds Barrangou, R. & van der Oost, J.) (Springer, 2013).
Makarova, K. S. et al. Evolution and classification of the CRISPR–Cas systems. Nat. Rev. Microbiol. 9, 467–477 (2011).
Makarova, K. S., Wolf, Y. I. & Koonin, E. V. Comparative genomics of defense systems in archaea and bacteria. Nucleic Acids Res. 41, 4360–4377 (2013).
Nuñez, J. K., Harrington, L. B., Kranzusch, P. J., Engelman, A. N. & Doudna, J. A. Foreign DNA capture during CRISPR–Cas adaptive immunity. Nature 527, 535–538 (2015).
Wang, J. et al. Structural and mechanistic basis of PAM-dependent spacer acquisition in CRISPR-Cas systems. Cell 163, 840–853 (2015).
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).
Kim, T. Y., Shin, M., Huynh Thi Yen, L. & Kim, J. S. Crystal structure of Cas1 from Archaeoglobus fulgidus and characterization of its nucleolytic activity. Biochem. Biophys. Res. Commun. 441, 720–725 (2013).
Wiedenheft, B. et al. Structural basis for DNase activity of a conserved protein implicated in CRISPR-mediated genome defense. Structure 17, 904–912 (2009).
Beloglazova, N. et al. A novel family of sequence-specific endoribonucleases associated with the clustered regularly interspaced short palindromic repeats. J. Biol. Chem. 283, 20361–20371 (2008).
Nam, K. H. et al. Double-stranded endonuclease activity in Bacillus halodurans clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas2 protein. J. Biol. Chem. 287, 35943–35952 (2012).
Samai, P., Smith, P. & Shuman, S. Structure of a CRISPR-associated protein Cas2 from Desulfovibrio vulgaris. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 66, 1552–1556 (2010).
Deveau, H. et al. Phage response to CRISPR-encoded resistance in Streptococcus thermophilus. J. Bacteriol. 190, 1390–1400 (2008).
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).
Shah, S. A., Erdmann, S., Mojica, F. J. & Garrett, R. A. Protospacer recognition motifs: mixed identities and functional diversity. RNA Biol. 10, 891–899 (2013).
Heler, R., Marraffini, L. A. & Bikard, D. Adapting to new threats: the generation of memory by CRISPR-Cas immune systems. Mol. Microbiol. 93, 1–9 (2014).
Levy, A. et al. CRISPR adaptation biases explain preference for acquisition of foreign DNA. Nature 520, 505–510 (2015).
Dillingham, M. S. & Kowalczykowski, S. C. RecBCD enzyme and the repair of double-stranded DNA breaks. Microbiol. Mol. Biol. Rev. 72, 642–671 (2008).
Stern, A., Keren, L., Wurtzel, O., Amitai, G. & Sorek, R. Self-targeting by CRISPR: gene regulation or autoimmunity? Trends Genet. 26, 335–340 (2010).
Bobay, L. M., Touchon, M. & Rocha, E. P. Manipulating or superseding host recombination functions: a dilemma that shapes phage evolvability. PLoS Genet. 9, e1003825 (2013).
Kuzminov, A. Single-strand interruptions in replicating chromosomes cause double-strand breaks. Proc. Natl Acad. Sci. USA 98, 8241–8246 (2001).
Michel, B. et al. Rescue of arrested replication forks by homologous recombination. Proc. Natl Acad. Sci. USA 98, 8181–8188 (2001).
Shee, C. et al. Engineered proteins detect spontaneous DNA breakage in human and bacterial cells. eLife 2, e01222 (2013).
Zhang, J., Kasciukovic, T. & White, M. F. The CRISPR associated protein Cas4 Is a 5′ to 3′ DNA exonuclease with an iron-sulfur cluster. PLoS ONE 7, e47232 (2012).
Lemak, S. et al. The CRISPR-associated Cas4 protein Pcal_0546 from Pyrobaculum calidifontis contains a [2Fe-2S] cluster: crystal structure and nuclease activity. Nucleic Acids Res. 42, 11144–11155 (2014).
Goren, M. G., Yosef, I., Auster, O. & Qimron, U. Experimental definition of a clustered regularly interspaced short palindromic duplicon in Escherichia coli. J. Mol. Biol. 423, 14–16 (2012).
Swarts, D. C., Mosterd, C., van Passel, M. W. & Brouns, S. J. CRISPR interference directs strand specific spacer acquisition. PLoS ONE 7, e35888 (2012).
Cote, A. G. & Lewis, S. M. Mus81-dependent double-strand DNA breaks at in vivo-generated cruciform structures in S. cerevisiae. Mol. Cell 31, 800–812 (2008).
Lilley, D. M. & White, M. F. The junction-resolving enzymes. Nat. Rev. Mol. Cell Biol. 2, 433–443 (2001).
Nam, K. H., Kurinov, I. & Ke, A. Crystal structure of clustered regularly interspaced short palindromic repeats (CRISPR)-associated Csn2 protein revealed Ca2+-dependent double-stranded DNA binding activity. J. Biol. Chem. 286, 30759–30768 (2011).
Makarova, K. S., Wolf, Y. I. & Koonin, E. V. The basic building blocks and evolution of CRISPR–CAS systems. Biochem. Soc. Trans. 41, 1392–1400 (2013).
Lemak, S. et al. Toroidal structure and DNA cleavage by the CRISPR-associated [4Fe-4S] cluster containing Cas4 nuclease SSO0001 from Sulfolobus solfataricus. J. Am. Chem. Soc. 135, 17476–17487 (2013).
Blosser, T. R. et al. Two distinct DNA binding modes guide dual roles of a CRISPR-Cas protein complex. Mol. Cell 58, 60–70 (2015).
Fineran, P. C. et al. Degenerate target sites mediate rapid primed CRISPR adaptation. Proc. Natl Acad. Sci. USA 111, E1629–E1638 (2014).
Richter, C. et al. Priming in the Type I-F CRISPR-Cas system triggers strand-independent spacer acquisition, bi-directionally from the primed protospacer. Nucleic Acids Res. 42, 8516–8526 (2014).
Krupovic, M., Makarova, K. S., Forterre, P., Prangishvili, D. & Koonin, E. V. Casposons: a new superfamily of self-synthesizing DNA transposons at the origin of prokaryotic CRISPR-Cas immunity. BMC Biol. 12, 36 (2014).
Koonin, E. V. & Krupovic, M. Evolution of adaptive immunity from transposable elements combined with innate immune systems. Nat. Rev. Genet. 16, 184–192 (2015).
Koonin, E. V. & Makarova, K. S. CRISPR-Cas: evolution of an RNA-based adaptive immunity system in prokaryotes. RNA Biol. 10, 679–686 (2013).
Paez-Espino, D. et al. Strong bias in the bacterial CRISPR elements that confer immunity to phage. Nat. Commun. 4, 1430 (2013).
Acknowledgements
The authors thank P. Fineran for discussions on primed acquisition. R.S. was supported, in part, by the Israel Science Foundation (personal grant 1303/12 and Israeli Centers of Research Excellence (I-CORE) grant 1796/12), the European Research Council (ERC; Starting Grant 260432), the Human Frontier Science Program (HFSP; grant RGP0011/2013), the Abisch-Frenkel foundation, the Pasteur-Weizmann council, the Institut Merieux, the Leona M. and Harry B. Helmsley Charitable Trust, the Minerva Foundation and the Deutsche Forschungsgemeinschaft (a Deutsch-Israelische Projektkooperation (DIP) grant).
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Amitai, G., Sorek, R. CRISPR–Cas adaptation: insights into the mechanism of action. Nat Rev Microbiol 14, 67–76 (2016). https://doi.org/10.1038/nrmicro.2015.14
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DOI: https://doi.org/10.1038/nrmicro.2015.14
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