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RNA Biology 9:5, 1-6; May 2012; © 2012 Landes Bioscience

The bacterial CRISPR/Cas system as analog of the mammalian adaptive immune system Moran G. Goren, Ido Yosef, Rotem Edgar and Udi Qimron* Department of Clinical Microbiology and Immunology; Sackler School of Medicine; Tel Aviv University; Tel Aviv, Israel

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acteria, like mammals, have to constantly defend themselves from viral attack. Like mammals, they use both innate and adaptive defense mechanisms. In this point of view we highlight the commonalities between defense systems of bacteria and mammals. Our focus is on the recently discovered bacterial adaptive immune system, the clustered regularly interspaced short palindromic repeats (CRISPR) and their associated proteins (Cas). We suggest that fundamental aspects of CRISPR/Cas immunity may be viewed in light of the vast accumulated knowledge on the mammalian immune system, and propose that further insights will be revealed by thorough comparison between the systems.

labeling the invading DNA as self.6,7 The restriction-modification defense system is semi-specific in the sense that many alien DNA molecules contain the recognition site. Some phages overcome this defense by eliminating the recognition site from their genome, or methylating it, or encoding a specific counter-defense element, such as a protein that inhibits the system.8-10 Another defense system used by certain bacteria is called “abortive infection.” This is an altruistic mechanism, in which an infected bacterium kills itself, thereby eliminating phage propagation. This mechanism is lethal to the single infected bacterium, but is evolutionarily conserved because it provides protection to the bacterial colony, comprised of individual bacteria sharing similar DNA.11 Recently, a novel type of defense system has been identified in bacteria: the CRISPR/Cas system.12 Unlike the other known defense systems, this system attacks nucleic acids having unique DNA sequences of ~20–50 bp. The system is encoded by DNA having a CRISPR array, leader sequence and cas genes.13-16 The CRISPR array is composed of short repetitive sequences, termed “repeats”, flanking similarly sized sequences termed “spacers”. The CRISPR array is transcribed and processed by Cas proteins to yield RNA spacers flanked by partial repeat sequences. The flanking repeats probably serve as “molecular handles” for recognition by the Cas proteins.14 Specific base-pairing between the processed spacers and alien nucleic acid results in elimination of the foreign nucleic acid, by one or more of the Cas proteins (in CRISPR type I, probably by Cas3 14,17,18). A unique feature of

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Key words: defense mechanism, innate immunity, chaperones Submitted: 02/28/12 Accepted: 03/27/12 http://dx.doi.org/10.4161/rna.20177 *Correspondence to: Udi Qimron; Email: ehudq@ post.tau.ac.il

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Bacteria are outnumbered by their viruses (phages) at an estimated ratio of 1:10.1 To survive in such a harsh environment, bacteria have developed various defense systems. Capsule formation is an efficient and common bacterial strategy to prevent phage attack.2-5 This mechanistic barrier prevents adsorption of phages and thus protects bacteria from infection. However, this mechanism is energetically expensive, both in production of the capsule and in the side effect of limiting diffusion of essential elements into the capsule-engulfed bacterium. Another type of defense is the restriction-modification system that recognizes short DNA sequences, called recognition sites, and cleaves incoming DNA containing them. Self-cleavage is prevented by specific methylation of hemimethylated DNA strands by a specific DNA methylase, which thus conserves the “self-identity” of host DNA without

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the CRISPR system is its ability to acquire new spacers from foreign nucleic acids, consequently arming the system against future invasions by the sampled nucleic acid. Thus, the CRISPR/Cas system is an adaptive immune system as it can adapt to defense against environmental aggressors by memorizing past invasions, recognizing them specifically, and initiating an efficient and specific response against them. The similarities and distinctions of the CRISPR/Cas system with that of the eukaryotic RNA interference pathway have been highlighted (reviewed in ref. 19–21). Here we highlight the analogies between the mammalian and prokaryotic immune systems, with a focus on the CRISPR/Cas system and the analogous adaptive immune system in mammals. The comparison is between concepts and principles of the two systems rather than between their detailed mechanisms of action. We show that fundamental questions, such as the advantages for bacteria of an adaptive immune system, can be addressed in light of these analogies.

complex 23,24), and this in turn causes certain players in the innate immune system, such as natural-killer cells, to kill the infected cells, thereby saving the entire organism. Self- or cell-mediated killing of virally infected mammalian cells can be compared in bacteria to abortive infection by a single bacterium as part of an entire bacterial colony’s rescue strategy. In addition to these innate defense mechanisms, mammals also have an adaptive defense system, which can memorize attackers, recognize them, and robustly respond to their subsequent invasion. For example, specialized memory B cells which have had a prior encounter with an antigen specifically multiply and secrete antibodies against that antigen, which can eventually lead to its neutralization.23 The analogous system in bacteria is the recently identified CRISPR/Cas system. This remarkable system is capable of mediating sequencespecific protection not only against invading phages and other harmful DNA,13,25 but also, as we have previously shown26 against prophages residing in the genome.

(crRNA) into smaller fragments analogous to antibodies, both of which confer specificity against the invader. It is interesting to note that in this respect, both antibodies and crRNAs have a constant region (Fc /partial flanking repeats) and a variable region (Fab /spacer), and these regions serve similar roles of recognition by the system components and recognition of the foreign element, respectively23,27 (Fig. 1). Naturally, antibodies can recognize complex structures because the recognition is based on the key-lock recognition mechanism, whereas the spacers recognize only nucleic acids via specific annealing of the two molecules. Nevertheless, the specificity and binding affinity of the two recognition modes are high in both cases. Certain regulatory proteins such as H-NS, LeuO, and the recently identified HtpG2832 are analogous to the T-helper cells, in the sense that they control the timing and strength of the response by regulating expression and steady-state levels of the effector proteins. For example, H-NS was shown in the E. coli CRISPR/Cas system to effectively shut down system activity by binding to the promoters regulating transcription of the system components.28,30 We have recently shown that in E. coli, HtpG acts as a positive modulator of the CRISPR/Cas system by increasing the steady-state level of an effector protein, Cas3.32 Cas3 is analogous to the cytotoxic T cells in that it probably specifically destroys DNA marked by crRNAs produced by the system.14,18 The analogy extends not only to components of the system, but also to similar principles of action. For example, the adaptive immune system shares similarities with the CRISPR/Cas system in terms of enhancing recognition efficiency of the foreign molecule. B cells undergo several cycles of recombination and mutagenesis in the antibody-encoding DNA region in response to antigen recognition, to optimize this recognition.23 Similarly, the acquisition of new spacers of a specific DNA molecule into the CRISPR array is stimulated by previous acquisition of spacers from the same foreign DNA, in a process termed “priming”. Apparently, the acquisition of new spacers is facilitated by the presence of a spacer against a specific phage genome. This facilitated adaptation

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Mammalian vs. Bacterial Defense Systems

Mammals, like other organisms, defend themselves against viruses and other infections by several mechanisms, which have analogous systems in bacteria. In general, the three types of defense mechanism are (1) physical barriers such as the skin, mucus and epithelial cells lining mucosal surfaces, (2) the innate immune system and (3) the adaptive immune system (Fig. 1). The skin layer and mucosal lining are physical barriers to most infections. In bacteria, the analogous physical barrier is the bacterial capsule, which prevents phages from entering the cell. If a pathogen does penetrate this line of defense, innate mechanisms such as neutrophils, macrophages and complement, immediately respond and attack the invader.22,23 The restriction-modification system in bacteria—their innate immune system—provides the second line of defense. Mammalian cells infected by a virus often display lower levels of certain molecules (such as class I major histocompatibility

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Analogous Components and Processes in the Mammalian and Bacterial Adaptive Immune Systems

The evolutionary function of the mammalian immune system is probably to keep the gonads functional for reproduction of the DNA that is shared by the organism’s different cell types. Similarly, in a bacterial colony, each member may be viewed as a “gonad,” and the evolutionary role of CRISPR/Cas and other bacterial defense systems is to protect their replication ability. In the mammalian system, specific cell lineages have evolved to play specific roles. In general, the cytotoxic T cells specifically recognize infected or cancerous cells and kill them, B cells constitute the antibody factory and T-helper cells orchestrate the responses by recruiting certain cells, secreting cytokines and determining response strength and type. The parallels for these different cell types in the CRISPR/ Cas system are particular proteins. For example, the CasABCDE protein complex in Escherichia coli is the equivalent of B cells, in the sense that it is responsible for processing the CRISPR-derived RNA

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Figure 1. Analogous components of the bacterial vs. mammalian defense systems.

increases the resistance against the phage as it becomes recognized more efficiently by additional spacers (Konstantin Severinov, personal communication). Both systems depend on the innate defense system in that disposal of the “debris” from the activity of the adaptive systems is performed by the innate mechanisms: in mammals, macrophages

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and other cells “clean up” the remnants, and in bacteria, motored exonucleases such as RecBCD presumably degrade the linearized DNA molecules cleaved by the system. This presumption is supported indirectly in that the core protein Cas4 is a RecB-like exonuclease,33 and in systems lacking Cas4, such as the E. coli system, another core Cas protein, Cas1, is shown

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to physically interact with both RecB and RecC.34 The adaptive immune system in mammals collaborates closely with the innate immune system. Cells of the latter system recognize foreign molecules and subsequently secrete cytokines, which in turn activate the former.23 This raises the possibility that such a regulatory interplay

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between the innate and adaptive systems also exists in bacteria. It is tempting to suggest that the initial exposure to a phage leading to the first acquisition event against that phage does not result in lethal infection because innate defense systems cleave the phage DNA, and this cleavage, in turn, may activate the adaptation machinery of the CRISPR/Cas. These speculations await experimental elucidation. The two systems also resemble each other in that both lose their activity against an invading element in the absence of a stimulus. The mammalian immune system loses memory cells in the absence of a stimulus from a specific antigen for a prolonged time. For example, a prolonged period without exposure to tetanus toxoid results in loss of the specific memory B cells producing the antibodies against tetanus toxin.23 Likewise, “old” spacers are constantly deleted from the pool of spacers if there is no phage challenge maintaining the selection pressure to preserve them.25,35 One major difference between the two systems is the initial state of the adaptive immune system. To the best of our knowledge, a newborn mammal does not inherit its adaptive immune state from its parents; rather, this system evolves from a “tabula rasa” state upon encounters with antigens.23 In contrast, a bacterial cell that is starting to form a colony has a memory of past invasions encountered by its ancestors in its CRISPR array, and is thus protected against them. In the new environment, it may encounter novel aggressors whose genomes may eventually be sampled into the CRISPR array and consequently inherited. Evidence of this Lamarckian type of immune inheritance in eukaryotes is also starting to emerge (e.g., Caenorhabditis elegans which has developed immunity against a specific virus passes this immunity on to its progeny36) and this may eventually prove to be a phenomenon that was first shown for the prokaryotic CRISPR/Cas, highlighting its occurrence in higher organisms.

system thoroughly may shed light on the other system since they share similar principles. Many universal systems have been elucidated first in lower organisms and later in higher organisms (e.g., the genetic code, DNA as the genetic information molecule, etc.). The bacterial CRISPR/ Cas system, however, has been much less studied than the mammalian adaptive immune system, and therefore principles in the mammalian system could reflect on the bacterial system. Indeed, key questions from known concepts in mammalian adaptive immunity have been addressed. For example, a classical question taken from mammalian immunity of whether the CRISPR/Cas system encounters autoimmunity was elegantly addressed for the bacterial system by Sorek and coworkers.37 They highlighted the fact that many spacers in bacterial CRISPR arrays contain sequences matching their own genome. Subsequent bioinformatics analyses revealed that such cases most often lead to loss of CRISPR/Cas functionality due to autoimmunity. Loss of functionality of the CRISPR/Cas system occurred via loss of cas genes, loss of the specific spacer or via other mechanisms.37 Another example is of a related concept, the recognition of self vs. foreign by the CRISPR/Cas system. This discrimination in the mammalian system is based on “training” the system components at a very early developmental stage to ignore any self-molecule that is present in the body. The parallel step in the CRISPR/ Cas system, adaptation, is still not well understood, and one might therefore wonder, along these lines, if there is a mechanism that allows the bacterium to discriminate its own DNA from the foreign one. This mechanism may require the “adaptation enzymes,” Cas1 and Cas2,38 to scan the DNA prior to sampling spacers from it, and perhaps fall off when they encounter sequences marking the DNA as “self.” The question of how the CRISPR array itself is recognized as self and not attacked was addressed by Marraffini and Sontheimer,24 who revealed that the flanking repeats, which remain partially intact in the processed crRNA, prevent such an attack. In addition, the requirement for an adjacent motif in the targeted DNA which is

absent from the CRISPR array also prevents such an attack.27 These examples show that important questions taken from the mammalian adaptive system should be considered in studying the CRISPR/Cas system, and that using this knowledge can guide us to solve some of the enigmas in the bacterial system. Requirement of Adaptive Defense against Specific DNA in Bacteria A fundamental question regarding the CRISPR/Cas system is why does a bacterium require adaptive defense against specific DNAs? Why are innate mechanisms, such as restriction enzymes, not sufficient? The rationale for the need for specificity in mammalian systems is that specificity allows robust responses with minimal collateral damage introduced by non-specific responses. For example, neutrophils can secrete reactive oxygen species against invading pathogens, but this may also harm the surrounding tissues, whereas antibody activity presents minimal collateral damage. This argument may also hold in innate systems of bacterial defense. For example, enhancing the restriction enzyme response may result in self-targeting due to the inability of the methylation system to “catch up” with the pace of DNA cleavage. Increasing the efficiency of methylase, in turn, might result in methylation of the incoming DNA, with the undesired outcome of protecting foreign DNA. In CRISPR/Cas, the response can be robust without the fear of self-cleavage, as it is targeted against sequences that are not present in the host genome. Another explanation for the need for specificity in the mammalian immune system lies in its essential activity of targeting virus-infected cells of the body or cancerous cells. These cells are marked by peptides displayed on specific receptors, and are killed by cytotoxic T cells.23 In this case specific recognition is a requirement for a response, and without it, suitable resolution is impossible. This explanation can be paralleled to the CRISPR/Cas system acting on lysogens integrated in the DNA genome.26 In this case, the only known mechanism in the bacterium that can act is the CRISPR/

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Autoimmunity in the CRISPR/Cas System The analogy between the distant systems, which reached optimal solutions independently, suggests that understanding one

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Cas system, by specific targeting of the self genome. On the one hand, this results in cell death, just like the killing of mammalian infected cells; on the other, it rescues the entire colony. Along the same lines, it has been suggested that a significant role of the CRISPR/Cas system is to eliminate extrachromosomal DNA encoding stress-inducing proteins.39 The observations leading to this hypothesis were that membrane stress caused by a plasmidencoded protein results in elimination of the plasmid, in a CRISPR/Cas-dependent manner, unless the DnaK chaperone alleviates the stress. Our lab recently showed that another chaperone, HtpG, is essential for activity of the E. coli CRISPR/Cas system.32 In its absence, the steady-state concentration of Cas3 in the cell is maintained at a non-functional level. This highlights HtpG’s role as a helper molecule to the effector molecule, just like T-helper cells activate the cytotoxic T cells. It is interesting to note that transcription of htpG, as well as of casD, casE and cas2, is regulated by the stress-induced transcription factor σ32, suggesting that indeed, the CRISPR/ Cas system may eliminate extrachromosomal elements under stress. This activity is only possible for an adaptive defense system which can specifically discriminate between DNAs, and not for innate mechanisms lacking this ability. One might claim that specificity of the CRISPR/Cas system is required not as the sole means of excluding viral DNA, but rather to allow selective import of beneficial DNA without being virally infected. In bacteria, horizontal gene transfer (HGT) is a major means of diversifying and increasing their DNA repertoire, and it is therefore possible that the specific responses against undesired DNA have evolved to allow a high frequency of HGT with minimal damage. A side effect of this selectivity for foreign DNA may have evolved into a defense system, which eventually began functioning as such. The fact that the CRISPR/Cas system is not essential and is found in only 40% of bacteria is in line with this claim, whereas in mammals, HGT is not a major means of acquiring DNA variation, and the adaptive immune system is found in all organisms as an essential component of the defense machinery.

Concluding Remarks We suggest that a comparison of the bacterial and mammalian defense systems, in particular their adaptive defense branches, may, in some cases, lead to a deeper understanding of the recently identified CRISPR/Cas system, whose mechanism of action and overall function are still elusive. We propose that questions such as recognition of self vs. foreign discrimination in the adaptation step be addressed with a view of the principles of the mammalian system. Other questions, such as regulation of the system, may also be addressed in light of the complexity observed in the function of the T-helper cells in the mammalian immune system. If the principles of the two systems indeed prove to be universal, then implications from one system should be valid for the other, and therefore novel insights into CRISPR/Cas may reveal mechanisms reflected in the mammalian immune system as well.

7. Tock MR, Dryden DT. The biology of restriction and anti-restriction. Curr Opin Microbiol 2005; 8:46672; PMID:15979932; http://dx.doi.org/10.1016/j. mib.2005.06.003. 8. Studier FW. Gene 0.3 of bacteriophage T7 acts to overcome the DNA restriction system of the host. J Mol Biol 1975; 94:283-95; PMID:1095770; http:// dx.doi.org/10.1016/0022-2836(75)90083-2. 9. Rifat D, Wright NT, Varney KM, Weber DJ, Black LW. Restriction endonuclease inhibitor IPI* of bacteriophage T4: a novel structure for a dedicated target. J Mol Biol 2008; 375:720-34; PMID:18037438; http://dx.doi.org/10.1016/j.jmb.2007.10.064. 10. Molineux IJ. In Abedon ST, Calendar RL, (Eds.) The Bacteriophages. Oxford University Press, Oxford 2005; 275-99. 11. Chopin MC, Chopin A, Bidnenko E. Phage abortive infection in lactococci: variations on a theme. Curr Opin Microbiol 2005; 8:473-9; PMID:15979388; http://dx.doi.org/10.1016/j.mib.2005.06.006. 12. Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science 2007; 315:1709-12; PMID:17379808; http://dx.doi.org/10.1126/science.1138140. 13. Deveau H, Garneau JE, Moineau S. CRISPR/ Cas system and its role in phage-bacteria interactions. Annu Rev Microbiol 2010; 64:475-93; PMID :20528693 ; http://dx.doi.org/10.1146/ annurev.micro.112408.134123. 14. Brouns SJ, Jore MM, Lundgren M, Westra ER, Slijkhuis RJ, Snijders AP, et al. Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 2008; 321:960-4; PMID:18703739; http://dx.doi. org/10.1126/science.1159689. 15. Marraffini LA, Sontheimer EJ. CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA. Science 2008; 322:1843-5; PMID:19095942; http://dx.doi.org/10.1126/science.1165771. 16. Haft DH, Selengut J, Mongodin EF, Nelson KE. A guild of 45 CRISPR-associated (Cas) protein families and multiple CRISPR/Cas subtypes exist in prokaryotic genomes. PLoS Comput Biol 2005; 1:60; PMID:16292354; http://dx.doi.org/10.1371/journal. pcbi.0010060. 17. Makarova KS, Haft DH, Barrangou R, Brouns SJ, Charpentier E, Horvath P, et al. Evolution and classification of the CRISPR-Cas systems. Nat Rev Microbiol 2011; 9:467-77; PMID:21552286; http:// dx.doi.org/10.1038/nrmicro2577. 18. Beloglazova N, Petit P, Flick R, Brown G, Savchenko A, Yakunin AF. Structure and activity of the Cas3 HD nuclease MJ0384, an effector enzyme of the CRISPR interference. EMBO J 2011; 30:461627; PMID:22009198; http://dx.doi.org/10.1038/ emboj.2011.377. 19. Sorek R, Kunin V, Hugenholtz P. CRISPR—a widespread system that provides acquired resistance against phages in bacteria and archaea. Nat Rev Microbiol 2008; 6:181-6; PMID:18157154; http:// dx.doi.org/10.1038/nrmicro1793. 20. Makarova KS, Aravind L, Grishin NV, Rogozin IB, Koonin EV. A DNA repair system specific for thermophilicArchaea and bacteria predicted by genomic context analysis. Nucleic Acids Res 2002; 30:48296; PMID:11788711; http://dx.doi.org/10.1093/ nar/30.2.482. 21. Wiedenheft B, Sternberg SH, Doudna JA. RNAguided genetic silencing systems in bacteria and archaea. Nature 2012; 482:331-8; PMID:22337052; http://dx.doi.org/10.1038/nature10886. 22. Medzhitov R. Recognition of microorganisms and activation of the immune response. Nature 2007; 449:819-26; PMID:17943118; http://dx.doi. org/10.1038/nature06246. 23. Abbas AK, Lichtman AH, Pillai S. Cellular and Molecular Immunology 2007; 6.

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We thank Ariel Munitz for critical reading of the manuscript and Camille Vainstein for professional language editing. Financial Support

U.Q. was supported by the Israel Science Foundation [611/10]; the Binational Science Foundation [2009218]; and a Marie Curie International Reintegration Grant [PIRG-GA-2009-256340]. References 1. Bergh O, Børsheim KY, Bratbak G, Heldal M. High abundance of viruses found in aquatic environments. Nature 1989; 340:467-8; PMID:2755508; http:// dx.doi.org/10.1038/340467a0. 2. Scholl D, Adhya S, Merril C. Escherichia coli K1’s capsule is a barrier to bacteriophage T7. Appl Environ Microbiol 2005; 71:4872-4; PMID:16085886; http://dx.doi.org/10.1128/AEM.71.8.4872-4.2005. 3. Qimron U, Marintcheva B, Tabor S, Richardson CC. Genomewide screens for Escherichia coli genes affecting growth of T7 bacteriophage. Proc Natl Acad Sci USA 2006; 103:19039-44; PMID:17135349; http:// dx.doi.org/10.1073/pnas.0609428103. 4. Camprubí S, Merino S, Benedí VJ, Tomás JM. Isolation and characterization of bacteriophage FC3-10 from Klebsiella spp. FEMS Microbiol Lett 1991; 67:291-7; PMID:1769536; http://dx.doi. org/10.1016/0378-1097(91)90491-R. 5. Bernheimer HP, Tiraby JG. Inhibition of phage infection by pneumococcus capsule. Virology 1976; 73:308-9; PMID:8868; http://dx.doi. org/10.1016/0042-6822(76)90085-4. 6. Bickle TA, Krüger DH. Biology of DNA restriction. Microbiol Rev 1993; 57:434-50; PMID:8336674.

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24. Storkus WJ, Dawson JR. Target structures involved in natural killing (NK): characteristics, distribution and candidate molecules. Crit Rev Immunol 1991; 10:393-416; PMID:2021423. 25. Bhaya D, Davison M, Barrangou R. CRISPR-Cas systems in bacteria and archaea: versatile small RNAs for adaptive defense and regulation. Annu Rev Genet 2011; 45:273-97; PMID:22060043; http://dx.doi. org/10.1146/annurev-genet-110410-132430. 26. Edgar R, Qimron U. The Escherichia coli CRISPR system protects from λ lysogenization, lysogens and prophage induction. J Bacteriol 2010; 192:62914; PMID:20889749; http://dx.doi.org/10.1128/ JB.00644-10. 27. Marraffini LA, Sontheimer EJ. Self versus non-self discrimination during CRISPR RNA-directed immunity. Nature 2010; 463:568-71; PMID:20072129; http://dx.doi.org/10.1038/nature08703. 28. Pul U, Wurm R, Arslan Z, Geissen R, Hofmann N, Wagner R. Identification and characterization of E. coli CRISPR-cas promoters and their silencing by H-NS. Mol Microbiol 2010; 75:1495-512; PMID:20132443; http://dx.doi.org/10.1111/j.13652958.2010.07073.x. 29. Medina-Aparicio L, Rebollar-Flores JE, GallegoHernández AL, VázquezA, Olvera L, GutiérrezRíos RM, et al. The CRISPR/Cas immune system is an operon regulated by LeuO, H-NS and leucine-responsive regulatory protein in Salmonella enterica serovar typhi. J Bacteriol 2011; 193:2396407; PMID:21398529; http://dx.doi.org/10.1128/ JB.01480-10.

30. Pougach K, Semenova E, Bogdanova E, Datsenko KA, Djordjevic M, Wanner BL, et al. Transcription, processing and function of CRISPR cassettes in Escherichia coli. MolMicrobiol 2010; 77:1367-79; PMID:20624226; http://dx.doi.org/10.1111/j.13652958.2010.07265.x. 31. Westra ER, Pul U, Heidrich N, Jore MM, Lundgren M, Stratmann T, et al. H-NS-mediated repression of CRISPR-based immunity in Escherichia coli K12 can be relieved by the transcription activator LeuO. Mol Microbiol 2010; 77:1380-93; PMID:20659289; http://dx.doi.org/10.1111/j.1365-2958.2010.07315.x. 32. Yosef I, Goren MG, Kiro R, Edgar R, Qimron U. High-temperature protein G is essential for activity of the Escherichia coli clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system. Proc Natl Acad Sci USA 2011; 108:2013641; PMID:22114197; http://dx.doi.org/10.1073/ pnas.1113519108. 33. Jansen R, Embden JD, Gaastra W, Schouls LM. Identification of genes that are associated with DNA repeats in prokaryotes. Mol Microbiol 2002; 43:1565-75; PMID:11952905; http://dx.doi. org/10.1046/j.1365-2958.2002.02839.x. 34. Babu M, Beloglazova N, Flick R, Graham C, Skarina T, Nocek B, et al. A dual function of the CRISPRCas system in bacterial antivirus immunity and DNA repair. Mol Microbiol 2011; 79:484-502; PMID:21219465; http://dx.doi.org/10.1111/j.13652958.2010.07465.x.

35. Deveau H, Barrangou R, Garneau JE, Labonté J, Fremaux C, Boyaval P, et al. Phage response to CRISPR-encoded resistance in Streptococcus thermophilus. J Bacteriol 2008; 190:1390-400; PMID :18065545; http://dx.doi.org/10.1128/ JB.01412-07. 36. Rechavi O, Minevich G, Hobert O. Transgenerational inheritance of an acquired small RNA-based antiviral response in C. elegans. Cell 2011; 147:124856; PMID:22119442; http://dx.doi.org/10.1016/j. cell.2011.10.042. 37. Stern A, Keren L, Wurtzel O, Amitai G, Sorek R. Self-targeting by CRISPR: gene regulation or autoimmunity? Trends Genet 2010; 26:335-40; PMID:20598393; http://dx.doi.org/10.1016/j. tig.2010.05.008. 38. Yosef I, Goren MG, Qimron U. Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli. Nucleic Acids Res 2012; In press; PMID:22402487; http://dx.doi.org/10.1093/nar/ gks216. 39. Perez-Rodriguez R, Haitjema C, Huang Q, Nam KH, Bernardis S, Ke A, et al. Envelope stress is a trigger of CRISPR RNA-mediated DNA silencing in Escherichia coli. Mol Microbiol 2011; 79:584-99; PMID:21255106; http://dx.doi.org/10.1111/j.13652958.2010.07482.x.

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