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CRISPR-Cas: Outstanding questions remain
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Comment
CRISPR-Cas: Outstanding questions remain Comment on “Diversity, evolution, and therapeutic applications of small RNAs in prokaryotic and eukaryotic immune systems” by Edwin L. Cooper and Nicola Overstreet Konstantin Severinov a,b,∗ a Skolkovo Institute of Science and Technology, Moscow, Russia b St. Petersburg State Polytechnical University, St. Petersburg, Russia
Received 6 December 2013; accepted 10 December 2013
Communicated by M. Frank-Kamenetskii
Cooper and Overstreet [1] provide a comprehensive review of immune functions of small RNA-based systems in pro- and eukaryotes. The review is largely based on a recent avalanche of data on prokaryotic CRISPR-Cas systems, a burgeoning field that has been generously—perhaps, too generously—reviewed before (see Refs. [2–9] for an incomplete list of excellent reviews). The recent realization of potential tremendous utility of CRISPR-Cas systems for genetic medicine is, of course, a most welcome development. However, in a way it may detract from very important fundamental questions about the biological role of CRISPR-Cas (restriction–modification systems have suffered a similar fate). Cooper and Overstreet [1] state in the Abstract of their review that “CRISPR-Cas system confers resistance to exogenous genetic elements such as phages and plasmids by allowing for the recognition and silencing of these genetic elements. Moreover, CRISPR-Cas serves as a memory of past exposures.” While correct, these statements imply that CRISPR systems’ raison d’etre is to provide immunity. However, while there is no doubt that CRISPR systems can provide immunity, this does not have to be their sole or even primary function at least in some systems. The case of Escherichia coli, arguably the best-understood and most-studied living organism, demonstrates this quite clearly. Most E. coli strains have a CRISPR system with a full complement of functional cas genes and, usually, two CRISPR cassettes. There is a tremendous variety of E. coli CRISPR alleles (defined as ordered sets of spacers in a cassette), which, one has to assume, reflects the different “recorded memories” of encounters of present-day strains ancestors with mobile genetic elements. There are several problems with such a view. First, identical CRISPR alleles are routinely recovered from geographically distant sites [10,11]. This implies that the shared history of phage encounters for strains harboring these alleles occurred prior to their global geographical dispersal or that the planet-wide redistribution of E. coli occurs much faster than the time a phage needs to leave its mark in CRISPR alleles of local population. The second curious observation is that among almost two thousand distinct E. coli spacers currently known, very few match known E. coli phage genomes. In other words, if the initial bioinformatic analyses that showed the more DOI of original article: http://dx.doi.org/10.1016/j.plrev.2013.11.002. * Correspondence to: Skolkovo Institute of Science and Technology, Moscow, Russia.
E-mail address: [email protected]. 1571-0645/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.plrev.2013.12.001
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K. Severinov / Physics of Life Reviews ••• (••••) •••–•••
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than expected frequency of phage- and plasmid-derived sequences in CRISPR cassette spacers [12,13] were done with E. coli, it would have been impossible to predict the immune function of CRISPR-Cas systems. The lack of phage-derived E. coli spacers is not due to the lack of E. coli phages known (there are plenty). Further, known E. coli phages are readily targeted by its CRISPR-Cas system when appropriate spacers are introduced by means of genetic engineering [14,15]. Thus, E. coli can target its known phages. It just “chooses” not to do so. One can speculate that E. coli spacers are derived from phages, which have not been isolated yet. Statistical analysis of the extent of spacer matching to known phages leads to inferences about the total size of the E. coli ‘phageome’ (distinct phage genomes infecting a host) that is extremely large (exceeding the known E. coli ‘phageome’ by almost two orders of magnitude, M.S. Gelfand; personal communication). The predicted existence of a very large number of novel E. coli phages does not match well the fact that it is hard to isolate a new (i.e., different from the ones already known) E. coli phage from the environment. We are thus left with a question: If CRISPR-Cas serves for defense, then what invisible genetic invaders the present-day E. coli are engaged in a fight with? Another fundamental question about the CRISPR-Cas function concerns the pseudo-Lamarcian process of new spacers acquisition. Obviously, care must be taken not to acquire a spacer from bacteria’s own DNA, for this would lead to self-destruction. Therefore, spacer acquisition machinery must either specifically target invading DNA, poorly recognize host DNA, or both. In the case of restriction–modification systems this problem is solved by the presence of an epigenetic mark (methylation) on “self” DNA that is absent in the invading “non-self” DNA. We know of no such marks recognized by CRISPR-Cas systems. This implies that unlike restriction–modification systems, which function continuously and are thus always ready to mount a response against an invader, CRISPR-Cas systems should be inhibited most of the time to prevent accidental acquisition of a host-derived spacer followed by auto-immunity and death. Upon phage infection of a naïve (i.e., lacking a spacer targeting phage DNA) host, CRISPR-Cas system should be strongly and rapidly activated to provide a chance for the cell to pick up a phage-derived spacer and then mount a protective response. Since phage genomes are much smaller than those of the host, targeting the acquisition machinery to the virus must be a very pressing issue. What signal(s) activates host CRISPR-Cas upon infection and how is the “right” choice of DNA that becomes a donor of protective spacer made is presently unclear. One should also bare in mind that an infected cell that manages, against all odds, to pick up a spacer from the phage will be rushing against the time as phages develop fast and replicate their DNA rapidly (which in itself should make it more difficult for CRISPR-Cas system to counter the infection). Moreover, most lytic phages have evolved strategies to kill the infecting cell early in infection. Therefore, even if an infected cell draws a “lucky” spacer and decreases the yield of progeny phage, benefiting the entire bacterial population, it may not be able to pass this useful trait to its own progeny. The complications mentioned above (and others that are not mentioned but are not less serious) make CRISPRCas look like a rather cumbersome and ineffective tool to control bacteriophage infections, especially infections by lytic phages. Other, much more efficient tools exist for this purpose. This realization should not deter from the fact that CRISPR-Cas systems are absolutely fascinating from the point of view of mechanistic molecular biology and, if anything, makes it even more interesting to determine the biological function(s) of these ubiquitous prokaryotic systems. Acknowledgement My research is supported by the Ministry of Education and Science of Russian Federation, project 14.B25.31.0004. References [1] Cooper EL, Overstreet N. Diversity, evolution, and therapeutic applications of small RNAs in prokaryotic and eukaryotic immune systems. Phys Life Rev 2013. http://dx.doi.org/10.1016/j.plrev.2013.11.002 [in this issue]. [2] Karginov FV, Hannon GJ. The CRISPR system: small RNA-guided defense in bacteria and archaea. Mol Cell 2010;37:7–19. [3] Deveau H, Garneau JE, Moineau S. CRISPR/Cas system and its role in phage–bacteria interactions. Annu Rev Microbiol 2010;64:475–93. [4] 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. [5] Wiedenheft B, Sternberg SH, Doudna JA. RNA-guided genetic silencing systems in bacteria and archaea. Nature 2012;482:331–8. [6] Barrangou R, Horvath P. CRISPR: new horizons in phage resistance and strain identification. Annu Rev Food Sci Technol 2012;3:143–62. [7] Bikard D, Marraffini LA. Innate and adaptive immunity in bacteria: mechanisms of programmed genetic variation to fight bacteriophages. Curr Opin Immunol 2012;24:15–20.
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[8] Barrangou R. CRISPR-Cas systems and RNA-guided interference. Wiley Interdiscip Rev RNA 2013;4:267–78. [9] Sorek R, Lawrence CM, Wiedenheft B. CRISPR-mediated adaptive immune systems in bacteria and archaea. Annu Rev Biochem 2013;82:237–66. [10] Díez-Villaseñor C, Almendros C, García-Martínez J, Mojica FJ. Diversity of CRISPR loci in Escherichia coli. Microbiology 2010;156:1351–61. [11] Touchon M, Rocha EP. The small, slow and specialized CRISPR and anti-CRISPR of Escherichia and Salmonella. PLoS ONE 2010;5:e11126. [12] Bolotin A, Quinquis B, Sorokin A, Ehrlich SD. Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology 2005;151:2551–61. [13] Mojica FJ, Diez-Villasenor C, Garcia-Martinez J, Soria E. Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J Mol Evol 2005;60:174–82. [14] Brouns SJ, Jore MM, Lundgren M, Westra ER, Slijkhuis RJ, Snijders AP, et al. CRISPR RNAs guide antiviral defense in prokaryotes. Science 2008;321:960–4. [15] Semenova E, Jore MM, Datsenko KA, Semenova A, Westra ER, Wanner BL, et al. A crRNA seed sequence governs CRISPR interference. Proc Natl Acad Sci USA 2011;108:10098–103.
[m3SC+; v 1.181; Prn:30/12/2013; 9:46] P.1 (1-3)
Available online at www.sciencedirect.com
ScienceDirect Physics of Life Reviews ••• (••••) •••–••• www.elsevier.com/locate/plrev
Comment
CRISPR-Cas: Outstanding questions remain Comment on “Diversity, evolution, and therapeutic applications of small RNAs in prokaryotic and eukaryotic immune systems” by Edwin L. Cooper and Nicola Overstreet Konstantin Severinov a,b,∗ a Skolkovo Institute of Science and Technology, Moscow, Russia b St. Petersburg State Polytechnical University, St. Petersburg, Russia
Received 6 December 2013; accepted 10 December 2013
Communicated by M. Frank-Kamenetskii
Cooper and Overstreet [1] provide a comprehensive review of immune functions of small RNA-based systems in pro- and eukaryotes. The review is largely based on a recent avalanche of data on prokaryotic CRISPR-Cas systems, a burgeoning field that has been generously—perhaps, too generously—reviewed before (see Refs. [2–9] for an incomplete list of excellent reviews). The recent realization of potential tremendous utility of CRISPR-Cas systems for genetic medicine is, of course, a most welcome development. However, in a way it may detract from very important fundamental questions about the biological role of CRISPR-Cas (restriction–modification systems have suffered a similar fate). Cooper and Overstreet [1] state in the Abstract of their review that “CRISPR-Cas system confers resistance to exogenous genetic elements such as phages and plasmids by allowing for the recognition and silencing of these genetic elements. Moreover, CRISPR-Cas serves as a memory of past exposures.” While correct, these statements imply that CRISPR systems’ raison d’etre is to provide immunity. However, while there is no doubt that CRISPR systems can provide immunity, this does not have to be their sole or even primary function at least in some systems. The case of Escherichia coli, arguably the best-understood and most-studied living organism, demonstrates this quite clearly. Most E. coli strains have a CRISPR system with a full complement of functional cas genes and, usually, two CRISPR cassettes. There is a tremendous variety of E. coli CRISPR alleles (defined as ordered sets of spacers in a cassette), which, one has to assume, reflects the different “recorded memories” of encounters of present-day strains ancestors with mobile genetic elements. There are several problems with such a view. First, identical CRISPR alleles are routinely recovered from geographically distant sites [10,11]. This implies that the shared history of phage encounters for strains harboring these alleles occurred prior to their global geographical dispersal or that the planet-wide redistribution of E. coli occurs much faster than the time a phage needs to leave its mark in CRISPR alleles of local population. The second curious observation is that among almost two thousand distinct E. coli spacers currently known, very few match known E. coli phage genomes. In other words, if the initial bioinformatic analyses that showed the more DOI of original article: http://dx.doi.org/10.1016/j.plrev.2013.11.002. * Correspondence to: Skolkovo Institute of Science and Technology, Moscow, Russia.
E-mail address: [email protected]. 1571-0645/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.plrev.2013.12.001
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K. Severinov / Physics of Life Reviews ••• (••••) •••–•••
2
than expected frequency of phage- and plasmid-derived sequences in CRISPR cassette spacers [12,13] were done with E. coli, it would have been impossible to predict the immune function of CRISPR-Cas systems. The lack of phage-derived E. coli spacers is not due to the lack of E. coli phages known (there are plenty). Further, known E. coli phages are readily targeted by its CRISPR-Cas system when appropriate spacers are introduced by means of genetic engineering [14,15]. Thus, E. coli can target its known phages. It just “chooses” not to do so. One can speculate that E. coli spacers are derived from phages, which have not been isolated yet. Statistical analysis of the extent of spacer matching to known phages leads to inferences about the total size of the E. coli ‘phageome’ (distinct phage genomes infecting a host) that is extremely large (exceeding the known E. coli ‘phageome’ by almost two orders of magnitude, M.S. Gelfand; personal communication). The predicted existence of a very large number of novel E. coli phages does not match well the fact that it is hard to isolate a new (i.e., different from the ones already known) E. coli phage from the environment. We are thus left with a question: If CRISPR-Cas serves for defense, then what invisible genetic invaders the present-day E. coli are engaged in a fight with? Another fundamental question about the CRISPR-Cas function concerns the pseudo-Lamarcian process of new spacers acquisition. Obviously, care must be taken not to acquire a spacer from bacteria’s own DNA, for this would lead to self-destruction. Therefore, spacer acquisition machinery must either specifically target invading DNA, poorly recognize host DNA, or both. In the case of restriction–modification systems this problem is solved by the presence of an epigenetic mark (methylation) on “self” DNA that is absent in the invading “non-self” DNA. We know of no such marks recognized by CRISPR-Cas systems. This implies that unlike restriction–modification systems, which function continuously and are thus always ready to mount a response against an invader, CRISPR-Cas systems should be inhibited most of the time to prevent accidental acquisition of a host-derived spacer followed by auto-immunity and death. Upon phage infection of a naïve (i.e., lacking a spacer targeting phage DNA) host, CRISPR-Cas system should be strongly and rapidly activated to provide a chance for the cell to pick up a phage-derived spacer and then mount a protective response. Since phage genomes are much smaller than those of the host, targeting the acquisition machinery to the virus must be a very pressing issue. What signal(s) activates host CRISPR-Cas upon infection and how is the “right” choice of DNA that becomes a donor of protective spacer made is presently unclear. One should also bare in mind that an infected cell that manages, against all odds, to pick up a spacer from the phage will be rushing against the time as phages develop fast and replicate their DNA rapidly (which in itself should make it more difficult for CRISPR-Cas system to counter the infection). Moreover, most lytic phages have evolved strategies to kill the infecting cell early in infection. Therefore, even if an infected cell draws a “lucky” spacer and decreases the yield of progeny phage, benefiting the entire bacterial population, it may not be able to pass this useful trait to its own progeny. The complications mentioned above (and others that are not mentioned but are not less serious) make CRISPRCas look like a rather cumbersome and ineffective tool to control bacteriophage infections, especially infections by lytic phages. Other, much more efficient tools exist for this purpose. This realization should not deter from the fact that CRISPR-Cas systems are absolutely fascinating from the point of view of mechanistic molecular biology and, if anything, makes it even more interesting to determine the biological function(s) of these ubiquitous prokaryotic systems. Acknowledgement My research is supported by the Ministry of Education and Science of Russian Federation, project 14.B25.31.0004. References [1] Cooper EL, Overstreet N. Diversity, evolution, and therapeutic applications of small RNAs in prokaryotic and eukaryotic immune systems. Phys Life Rev 2013. http://dx.doi.org/10.1016/j.plrev.2013.11.002 [in this issue]. [2] Karginov FV, Hannon GJ. The CRISPR system: small RNA-guided defense in bacteria and archaea. Mol Cell 2010;37:7–19. [3] Deveau H, Garneau JE, Moineau S. CRISPR/Cas system and its role in phage–bacteria interactions. Annu Rev Microbiol 2010;64:475–93. [4] 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. [5] Wiedenheft B, Sternberg SH, Doudna JA. RNA-guided genetic silencing systems in bacteria and archaea. Nature 2012;482:331–8. [6] Barrangou R, Horvath P. CRISPR: new horizons in phage resistance and strain identification. Annu Rev Food Sci Technol 2012;3:143–62. [7] Bikard D, Marraffini LA. Innate and adaptive immunity in bacteria: mechanisms of programmed genetic variation to fight bacteriophages. Curr Opin Immunol 2012;24:15–20.
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K. Severinov / Physics of Life Reviews ••• (••••) •••–•••
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[8] Barrangou R. CRISPR-Cas systems and RNA-guided interference. Wiley Interdiscip Rev RNA 2013;4:267–78. [9] Sorek R, Lawrence CM, Wiedenheft B. CRISPR-mediated adaptive immune systems in bacteria and archaea. Annu Rev Biochem 2013;82:237–66. [10] Díez-Villaseñor C, Almendros C, García-Martínez J, Mojica FJ. Diversity of CRISPR loci in Escherichia coli. Microbiology 2010;156:1351–61. [11] Touchon M, Rocha EP. The small, slow and specialized CRISPR and anti-CRISPR of Escherichia and Salmonella. PLoS ONE 2010;5:e11126. [12] Bolotin A, Quinquis B, Sorokin A, Ehrlich SD. Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology 2005;151:2551–61. [13] Mojica FJ, Diez-Villasenor C, Garcia-Martinez J, Soria E. Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J Mol Evol 2005;60:174–82. [14] Brouns SJ, Jore MM, Lundgren M, Westra ER, Slijkhuis RJ, Snijders AP, et al. CRISPR RNAs guide antiviral defense in prokaryotes. Science 2008;321:960–4. [15] Semenova E, Jore MM, Datsenko KA, Semenova A, Westra ER, Wanner BL, et al. A crRNA seed sequence governs CRISPR interference. Proc Natl Acad Sci USA 2011;108:10098–103.