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1.  Diverse CRISPRs Evolving in Human Microbiomes 
PLoS Genetics  2012;8(6):e1002441.
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) loci, together with cas (CRISPR–associated) genes, form the CRISPR/Cas adaptive immune system, a primary defense strategy that eubacteria and archaea mobilize against foreign nucleic acids, including phages and conjugative plasmids. Short spacer sequences separated by the repeats are derived from foreign DNA and direct interference to future infections. The availability of hundreds of shotgun metagenomic datasets from the Human Microbiome Project (HMP) enables us to explore the distribution and diversity of known CRISPRs in human-associated microbial communities and to discover new CRISPRs. We propose a targeted assembly strategy to reconstruct CRISPR arrays, which whole-metagenome assemblies fail to identify. For each known CRISPR type (identified from reference genomes), we use its direct repeat consensus sequence to recruit reads from each HMP dataset and then assemble the recruited reads into CRISPR loci; the unique spacer sequences can then be extracted for analysis. We also identified novel CRISPRs or new CRISPR variants in contigs from whole-metagenome assemblies and used targeted assembly to more comprehensively identify these CRISPRs across samples. We observed that the distributions of CRISPRs (including 64 known and 86 novel ones) are largely body-site specific. We provide detailed analysis of several CRISPR loci, including novel CRISPRs. For example, known streptococcal CRISPRs were identified in most oral microbiomes, totaling ∼8,000 unique spacers: samples resampled from the same individual and oral site shared the most spacers; different oral sites from the same individual shared significantly fewer, while different individuals had almost no common spacers, indicating the impact of subtle niche differences on the evolution of CRISPR defenses. We further demonstrate potential applications of CRISPRs to the tracing of rare species and the virus exposure of individuals. This work indicates the importance of effective identification and characterization of CRISPR loci to the study of the dynamic ecology of microbiomes.
Author Summary
Human bodies are complex ecological systems in which various microbial organisms and viruses interact with each other and with the human host. The Human Microbiome Project (HMP) has resulted in >700 datasets of shotgun metagenomic sequences, from which we can learn about the compositions and functions of human-associated microbial communities. CRISPR/Cas systems are a widespread class of adaptive immune systems in bacteria and archaea, providing acquired immunity against foreign nucleic acids: CRISPR/Cas defense pathways involve integration of viral- or plasmid-derived DNA segments into CRISPR arrays (forming spacers between repeated structural sequences), and expression of short crRNAs from these single repeat-spacer units, to generate interference to future invading foreign genomes. Powered by an effective computational approach (the targeted assembly approach for CRISPR), our analysis of CRISPR arrays in the HMP datasets provides the very first global view of bacterial immunity systems in human-associated microbial communities. The great diversity of CRISPR spacers we observed among different body sites, in different individuals, and in single individuals over time, indicates the impact of subtle niche differences on the evolution of CRISPR defenses and indicates the key role of bacteriophage (and plasmids) in shaping human microbial communities.
doi:10.1371/journal.pgen.1002441
PMCID: PMC3374615  PMID: 22719260
2.  A PNPase Dependent CRISPR System in Listeria 
PLoS Genetics  2014;10(1):e1004065.
The human bacterial pathogen Listeria monocytogenes is emerging as a model organism to study RNA-mediated regulation in pathogenic bacteria. A class of non-coding RNAs called CRISPRs (clustered regularly interspaced short palindromic repeats) has been described to confer bacterial resistance against invading bacteriophages and conjugative plasmids. CRISPR function relies on the activity of CRISPR associated (cas) genes that encode a large family of proteins with nuclease or helicase activities and DNA and RNA binding domains. Here, we characterized a CRISPR element (RliB) that is expressed and processed in the L. monocytogenes strain EGD-e, which is completely devoid of cas genes. Structural probing revealed that RliB has an unexpected secondary structure comprising basepair interactions between the repeats and the adjacent spacers in place of canonical hairpins formed by the palindromic repeats. Moreover, in contrast to other CRISPR-Cas systems identified in Listeria, RliB-CRISPR is ubiquitously present among Listeria genomes at the same genomic locus and is never associated with the cas genes. We showed that RliB-CRISPR is a substrate for the endogenously encoded polynucleotide phosphorylase (PNPase) enzyme. The spacers of the different Listeria RliB-CRISPRs share many sequences with temperate and virulent phages. Furthermore, we show that a cas-less RliB-CRISPR lowers the acquisition frequency of a plasmid carrying the matching protospacer, provided that trans encoded cas genes of a second CRISPR-Cas system are present in the genome. Importantly, we show that PNPase is required for RliB-CRISPR mediated DNA interference. Altogether, our data reveal a yet undescribed CRISPR system whose both processing and activity depend on PNPase, highlighting a new and unexpected function for PNPase in “CRISPRology”.
Author Summary
CRISPR-Cas systems confer to bacteria and archaea an adaptive immunity that protects them against invading bacteriophages and plasmids. In this study, we characterize a CRISPR (RliB-CRISPR) that is present in all L. monocytogenes strains at the same genomic locus but is never associated with a cas operon. It is an unusual CRISPR that, as we demonstrate, has a secondary structure consisting of basepair interactions between the repeat sequence and the adjacent spacer. We show that the RliB-CRISPR is processed by the endogenously encoded polynucleotide phosphorylase enzyme (PNPase). In addition, we show that the RliB-CRISPR system requires PNPase and presence of trans encoded cas genes of a second CRISPR-Cas system, to mediate DNA interference directed against a plasmid carrying a matching protospacer. Altogether, our data reveal a novel type of CRISPR system in bacteria that requires endogenously encoded PNPase enzyme for its processing and interference activity.
doi:10.1371/journal.pgen.1004065
PMCID: PMC3886909  PMID: 24415952
3.  Diversity, Activity, and Evolution of CRISPR Loci in Streptococcus thermophilus▿ † 
Journal of Bacteriology  2007;190(4):1401-1412.
Clustered regularly interspaced short palindromic repeats (CRISPR) are hypervariable loci widely distributed in prokaryotes that provide acquired immunity against foreign genetic elements. Here, we characterize a novel Streptococcus thermophilus locus, CRISPR3, and experimentally demonstrate its ability to integrate novel spacers in response to bacteriophage. Also, we analyze CRISPR diversity and activity across three distinct CRISPR loci in several S. thermophilus strains. We show that both CRISPR repeats and cas genes are locus specific and functionally coupled. A total of 124 strains were studied, and 109 unique spacer arrangements were observed across the three CRISPR loci. Overall, 3,626 spacers were analyzed, including 2,829 for CRISPR1 (782 unique), 173 for CRISPR2 (16 unique), and 624 for CRISPR3 (154 unique). Sequence analysis of the spacers revealed homology and identity to phage sequences (77%), plasmid sequences (16%), and S. thermophilus chromosomal sequences (7%). Polymorphisms were observed for the CRISPR repeats, CRISPR spacers, cas genes, CRISPR motif, locus architecture, and specific sequence content. Interestingly, CRISPR loci evolved both via polarized addition of novel spacers after exposure to foreign genetic elements and via internal deletion of spacers. We hypothesize that the level of diversity is correlated with relative CRISPR activity and propose that the activity is highest for CRISPR1, followed by CRISPR3, while CRISPR2 may be degenerate. Globally, the dynamic nature of CRISPR loci might prove valuable for typing and comparative analyses of strains and microbial populations. Also, CRISPRs provide critical insights into the relationships between prokaryotes and their environments, notably the coevolution of host and viral genomes.
doi:10.1128/JB.01415-07
PMCID: PMC2238196  PMID: 18065539
4.  Persisting Viral Sequences Shape Microbial CRISPR-based Immunity 
PLoS Computational Biology  2012;8(4):e1002475.
Well-studied innate immune systems exist throughout bacteria and archaea, but a more recently discovered genomic locus may offer prokaryotes surprising immunological adaptability. Mediated by a cassette-like genomic locus termed Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), the microbial adaptive immune system differs from its eukaryotic immune analogues by incorporating new immunities unidirectionally. CRISPR thus stores genomically recoverable timelines of virus-host coevolution in natural organisms refractory to laboratory cultivation. Here we combined a population genetic mathematical model of CRISPR-virus coevolution with six years of metagenomic sequencing to link the recoverable genomic dynamics of CRISPR loci to the unknown population dynamics of virus and host in natural communities. Metagenomic reconstructions in an acid-mine drainage system document CRISPR loci conserving ancestral immune elements to the base-pair across thousands of microbial generations. This ‘trailer-end conservation’ occurs despite rapid viral mutation and despite rapid prokaryotic genomic deletion. The trailer-ends of many reconstructed CRISPR loci are also largely identical across a population. ‘Trailer-end clonality’ occurs despite predictions of host immunological diversity due to negative frequency dependent selection (kill the winner dynamics). Statistical clustering and model simulations explain this lack of diversity by capturing rapid selective sweeps by highly immune CRISPR lineages. Potentially explaining ‘trailer-end conservation,’ we record the first example of a viral bloom overwhelming a CRISPR system. The polyclonal viruses bloom even though they share sequences previously targeted by host CRISPR loci. Simulations show how increasing random genomic deletions in CRISPR loci purges immunological controls on long-lived viral sequences, allowing polyclonal viruses to bloom and depressing host fitness. Our results thus link documented patterns of genomic conservation in CRISPR loci to an evolutionary advantage against persistent viruses. By maintaining old immunities, selection may be tuning CRISPR-mediated immunity against viruses reemerging from lysogeny or migration.
Author Summary
Most microbes appear unculturable in the laboratory, limiting our knowledge of how virus and prokaryotic host evolve in natural systems. However, a genomic locus found in many prokaryotes, CRISPR, may offer cultivation-independent probes of virus-microbe coevolution. Utilizing nearby genes, CRISPR can serially incorporate short viral and plasmid sequences. These sequences bind and cleave cognate regions in subsequent viral and plasmid insertions, conferring adaptive anti-viral and anti-plasmid immunity. By incorporating sequences undirectionally, CRISPR also provides timelines of virus-prokaryote coevolution. Yet, CRISPR only incorporates 30–80 base-pair viral sequences, leaving incomplete coevolutionary recordings. To reconstruct the missing coevolutionary dynamics shaping natural CRISPRs, we combined metagenomic reconstructions with population-scale mathematical modeling. Capturing rare and rapid sweeps of CRISPR diversity by highly immune lines, mathematical modeling explains why naturally reconstructed CRISPR loci are often largely identical across a population. Both model and experiment further document surprising proliferations of old viral sequences against which hosts had preexisting CRISPR immunity. Due to these deadly blooms of ancestral viral elements, CRISPR's conservation of old immune sequences appears to confer a selective advantage. This may explain the striking immunological memory documented in CRISPR loci, which occurs despite rapid viral mutation and despite rapid deletions in prokaryotic genomes.
doi:10.1371/journal.pcbi.1002475
PMCID: PMC3330103  PMID: 22532794
5.  Cleavage of Phage DNA by the Streptococcus thermophilus CRISPR3-Cas System 
PLoS ONE  2012;7(7):e40913.
Streptococcus thermophilus, similar to other Bacteria and Archaea, has developed defense mechanisms to protect cells against invasion by foreign nucleic acids, such as virus infections and plasmid transformations. One defense system recently described in these organisms is the CRISPR-Cas system (Clustered Regularly Interspaced Short Palindromic Repeats loci coupled to CRISPR-associated genes). Two S. thermophilus CRISPR-Cas systems, CRISPR1-Cas and CRISPR3-Cas, have been shown to actively block phage infection. The CRISPR1-Cas system interferes by cleaving foreign dsDNA entering the cell in a length-specific and orientation-dependant manner. Here, we show that the S. thermophilus CRISPR3-Cas system acts by cleaving phage dsDNA genomes at the same specific position inside the targeted protospacer as observed with the CRISPR1-Cas system. Only one cleavage site was observed in all tested strains. Moreover, we observed that the CRISPR1-Cas and CRISPR3-Cas systems are compatible and, when both systems are present within the same cell, provide increased resistance against phage infection by both cleaving the invading dsDNA. We also determined that overall phage resistance efficiency is correlated to the total number of newly acquired spacers in both CRISPR loci.
doi:10.1371/journal.pone.0040913
PMCID: PMC3401199  PMID: 22911717
6.  A Guild of 45 CRISPR-Associated (Cas) Protein Families and Multiple CRISPR/Cas Subtypes Exist in Prokaryotic Genomes 
PLoS Computational Biology  2005;1(6):e60.
Clustered regularly interspaced short palindromic repeats (CRISPRs) are a family of DNA direct repeats found in many prokaryotic genomes. Repeats of 21–37 bp typically show weak dyad symmetry and are separated by regularly sized, nonrepetitive spacer sequences. Four CRISPR-associated (Cas) protein families, designated Cas1 to Cas4, are strictly associated with CRISPR elements and always occur near a repeat cluster. Some spacers originate from mobile genetic elements and are thought to confer “immunity” against the elements that harbor these sequences. In the present study, we have systematically investigated uncharacterized proteins encoded in the vicinity of these CRISPRs and found many additional protein families that are strictly associated with CRISPR loci across multiple prokaryotic species. Multiple sequence alignments and hidden Markov models have been built for 45 Cas protein families. These models identify family members with high sensitivity and selectivity and classify key regulators of development, DevR and DevS, in Myxococcus xanthus as Cas proteins. These identifications show that CRISPR/cas gene regions can be quite large, with up to 20 different, tandem-arranged cas genes next to a repeat cluster or filling the region between two repeat clusters. Distinctive subsets of the collection of Cas proteins recur in phylogenetically distant species and correlate with characteristic repeat periodicity. The analyses presented here support initial proposals of mobility of these units, along with the likelihood that loci of different subtypes interact with one another as well as with host cell defensive, replicative, and regulatory systems. It is evident from this analysis that CRISPR/cas loci are larger, more complex, and more heterogeneous than previously appreciated.
Synopsis
The family of clustered regularly interspaced short palindromic repeats (CRISPRs) describes a class of DNA repeats found in nearly half of all bacterial and archaeal genomes. These DNA repeat regions have a remarkably regular structure: unique sequences of constant size, called spacers, sit between each pair of repeats. The DNA repeats do not encode proteins, but appear to be transcribed and processed into small RNAs that may have any number of functions, including resistance to any phage (i.e., virus of bacteria) whose sequence matches a spacer; spacers change rapidly as microbial strains evolve. This work describes 41 new CRISPR-associated (cas) gene families, which are always found near these repeats, in addition to the four previously known. It shows that CRISPR systems belong to different classes, with different repeat patterns, sets of genes, and species ranges. Most of these seem to come and go rather rapidly from their host genomes. These possibly beneficial mobile genetic elements may play an important role in driving prokaryotic evolution.
doi:10.1371/journal.pcbi.0010060
PMCID: PMC1282333  PMID: 16292354
7.  The Small, Slow and Specialized CRISPR and Anti-CRISPR of Escherichia and Salmonella 
PLoS ONE  2010;5(6):e11126.
Prokaryotes thrive in spite of the vast number and diversity of their viruses. This partly results from the evolution of mechanisms to inactivate or silence the action of exogenous DNA. Among these, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) are unique in providing adaptive immunity against elements with high local resemblance to genomes of previously infecting agents. Here, we analyze the CRISPR loci of 51 complete genomes of Escherichia and Salmonella. CRISPR are in two pairs of loci in Escherichia, one single pair in Salmonella, each pair showing a similar turnover rate, repeat sequence and putative linkage to a common set of cas genes. Yet, phylogeny shows that CRISPR and associated cas genes have different evolutionary histories, the latter being frequently exchanged or lost. In our set, one CRISPR pair seems specialized in plasmids often matching genes coding for the replication, conjugation and antirestriction machinery. Strikingly, this pair also matches the cognate cas genes in which case these genes are absent. The unexpectedly high conservation of this anti-CRISPR suggests selection to counteract the invasion of mobile elements containing functional CRISPR/cas systems. There are few spacers in most CRISPR, which rarely match genomes of known phages. Furthermore, we found that strains divergent less than 250 thousand years ago show virtually identical CRISPR. The lack of congruence between cas, CRISPR and the species phylogeny and the slow pace of CRISPR change make CRISPR poor epidemiological markers in enterobacteria. All these observations are at odds with the expectedly abundant and dynamic repertoire of spacers in an immune system aiming at protecting bacteria from phages. Since we observe purifying selection for the maintenance of CRISPR these results suggest that alternative evolutionary roles for CRISPR remain to be uncovered.
doi:10.1371/journal.pone.0011126
PMCID: PMC2886076  PMID: 20559554
8.  Nasty Viruses, Costly Plasmids, Population Dynamics, and the Conditions for Establishing and Maintaining CRISPR-Mediated Adaptive Immunity in Bacteria 
PLoS Genetics  2010;6(10):e1001171.
Clustered, Regularly Interspaced Short Palindromic Repeats (CRISPR) abound in the genomes of almost all archaebacteria and nearly half the eubacteria sequenced. Through a genetic interference mechanism, bacteria with CRISPR regions carrying copies of the DNA of previously encountered phage and plasmids abort the replication of phage and plasmids with these sequences. Thus it would seem that protection against infecting phage and plasmids is the selection pressure responsible for establishing and maintaining CRISPR in bacterial populations. But is it? To address this question and provide a framework and hypotheses for the experimental study of the ecology and evolution of CRISPR, I use mathematical models of the population dynamics of CRISPR-encoding bacteria with lytic phage and conjugative plasmids. The results of the numerical (computer simulation) analysis of the properties of these models with parameters in the ranges estimated for Escherichia coli and its phage and conjugative plasmids indicate: (1) In the presence of lytic phage there are broad conditions where bacteria with CRISPR-mediated immunity will have an advantage in competition with non-CRISPR bacteria with otherwise higher Malthusian fitness. (2) These conditions for the existence of CRISPR are narrower when there is envelope resistance to the phage. (3) While there are situations where CRISPR-mediated immunity can provide bacteria an advantage in competition with higher Malthusian fitness bacteria bearing deleterious conjugative plasmids, the conditions for this to obtain are relatively narrow and the intensity of selection favoring CRISPR weak. The parameters of these models can be independently estimated, the assumption behind their construction validated, and the hypotheses generated from the analysis of their properties tested in experimental populations of bacteria with lytic phage and conjugative plasmids. I suggest protocols for estimating these parameters and outline the design of experiments to evaluate the validity of these models and test these hypotheses.
Author Summary
CRISPR is the acronym for the adaptive immune system that has been found in almost all archaebacteria and nearly half the eubacteria examined. Unlike the other defenses bacteria have for protection from phage and other deleterious DNAs, CRISPR has the virtues of specificity, memory, and the capacity to abort infections with a virtually indefinite diversity of deleterious DNAs. In this report, mathematical models of the population dynamics of bacteria, phage, and plasmids are used to determine the conditions under which CRISPR can become established and will be maintained in bacterial populations and the contribution of this adaptive immune system to the ecology and (co)evolution of bacteria and bacteriophage. The models predict realistic and broad conditions under which bacteria bearing CRISPR regions can invade and be maintained in populations of higher fitness bacteria confronted with bacteriophage and narrower conditions when the confrontation is with competitors carrying conjugative plasmids. The models predict that CRISPR can facilitate long-term co-evolutionary arms races between phage and bacteria and between phage- rather than resource-limited bacterial communities. The parameters of these models can be independently estimated, the assumptions behind their construction validated, and the hypotheses generated from the analysis of their properties tested with experimental populations of bacteria.
doi:10.1371/journal.pgen.1001171
PMCID: PMC2965746  PMID: 21060859
9.  CRISPR-Cas systems target a diverse collection of invasive mobile genetic elements in human microbiomes 
Genome Biology  2013;14(4):R40.
Background
Bacteria and archaea develop immunity against invading genomes by incorporating pieces of the invaders' sequences, called spacers, into a clustered regularly interspaced short palindromic repeats (CRISPR) locus between repeats, forming arrays of repeat-spacer units. When spacers are expressed, they direct CRISPR-associated (Cas) proteins to silence complementary invading DNA. In order to characterize the invaders of human microbiomes, we use spacers from CRISPR arrays that we had previously assembled from shotgun metagenomic datasets, and identify contigs that contain these spacers' targets.
Results
We discover 95,000 contigs that are putative invasive mobile genetic elements, some targeted by hundreds of CRISPR spacers. We find that oral sites in healthy human populations have a much greater variety of mobile genetic elements than stool samples. Mobile genetic elements carry genes encoding diverse functions: only 7% of the mobile genetic elements are similar to known phages or plasmids, although a much greater proportion contain phage- or plasmid-related genes. A small number of contigs share similarity with known integrative and conjugative elements, providing the first examples of CRISPR defenses against this class of element. We provide detailed analyses of a few large mobile genetic elements of various types, and a relative abundance analysis of mobile genetic elements and putative hosts, exploring the dynamic activities of mobile genetic elements in human microbiomes. A joint analysis of mobile genetic elements and CRISPRs shows that protospacer-adjacent motifs drive their interaction network; however, some CRISPR-Cas systems target mobile genetic elements lacking motifs.
Conclusions
We identify a large collection of invasive mobile genetic elements in human microbiomes, an important resource for further study of the interaction between the CRISPR-Cas immune system and invaders.
doi:10.1186/gb-2013-14-4-r40
PMCID: PMC4053933  PMID: 23628424
CRISPR-Cas system; human microbiome; mobile genetic element (MGE)
10.  Viral Diversity Threshold for Adaptive Immunity in Prokaryotes 
mBio  2012;3(6):e00456-12.
ABSTRACT
Bacteria and archaea face continual onslaughts of rapidly diversifying viruses and plasmids. Many prokaryotes maintain adaptive immune systems known as clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated genes (Cas). CRISPR-Cas systems are genomic sensors that serially acquire viral and plasmid DNA fragments (spacers) that are utilized to target and cleave matching viral and plasmid DNA in subsequent genomic invasions, offering critical immunological memory. Only 50% of sequenced bacteria possess CRISPR-Cas immunity, in contrast to over 90% of sequenced archaea. To probe why half of bacteria lack CRISPR-Cas immunity, we combined comparative genomics and mathematical modeling. Analysis of hundreds of diverse prokaryotic genomes shows that CRISPR-Cas systems are substantially more prevalent in thermophiles than in mesophiles. With sequenced bacteria disproportionately mesophilic and sequenced archaea mostly thermophilic, the presence of CRISPR-Cas appears to depend more on environmental temperature than on bacterial-archaeal taxonomy. Mutation rates are typically severalfold higher in mesophilic prokaryotes than in thermophilic prokaryotes. To quantitatively test whether accelerated viral mutation leads microbes to lose CRISPR-Cas systems, we developed a stochastic model of virus-CRISPR coevolution. The model competes CRISPR-Cas-positive (CRISPR-Cas+) prokaryotes against CRISPR-Cas-negative (CRISPR-Cas−) prokaryotes, continually weighing the antiviral benefits conferred by CRISPR-Cas immunity against its fitness costs. Tracking this cost-benefit analysis across parameter space reveals viral mutation rate thresholds beyond which CRISPR-Cas cannot provide sufficient immunity and is purged from host populations. These results offer a simple, testable viral diversity hypothesis to explain why mesophilic bacteria disproportionately lack CRISPR-Cas immunity. More generally, fundamental limits on the adaptability of biological sensors (Lamarckian evolution) are predicted.
IMPORTANCE
A remarkable recent discovery in microbiology is that bacteria and archaea possess systems conferring immunological memory and adaptive immunity. Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated genes (CRISPR-Cas) are genomic sensors that allow prokaryotes to acquire DNA fragments from invading viruses and plasmids. Providing immunological memory, these stored fragments destroy matching DNA in future viral and plasmid invasions. CRISPR-Cas systems also provide adaptive immunity, keeping up with mutating viruses and plasmids by continually acquiring new DNA fragments. Surprisingly, less than 50% of mesophilic bacteria, in contrast to almost 90% of thermophilic bacteria and Archaea, maintain CRISPR-Cas immunity. Using mathematical modeling, we probe this dichotomy, showing how increased viral mutation rates can explain the reduced prevalence of CRISPR-Cas systems in mesophiles. Rapidly mutating viruses outrun CRISPR-Cas immune systems, likely decreasing their prevalence in bacterial populations. Thus, viral adaptability may select against, rather than for, immune adaptability in prokaryotes.
doi:10.1128/mBio.00456-12
PMCID: PMC3517865  PMID: 23221803
11.  Self vs. non-self discrimination during CRISPR RNA-directed immunity 
Nature  2010;463(7280):568-571.
All immune systems must distinguish self from non-self to repel invaders without inducing autoimmunity. Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci protect bacteria and archaea from invasion by phage and plasmid DNA through a genetic interference pathway1–9. CRISPR loci are present in ~ 40% and ~90% of sequenced bacterial and archaeal genomes respectively10 and evolve rapidly, acquiring new spacer sequences to adapt to highly dynamic viral populations1, 11–13. Immunity requires a sequence match between the invasive DNA and the spacers that lie between CRISPR repeats1–9. Each cluster is genetically linked to a subset of the cas (CRISPR-associated) genes14–16 that collectively encode >40 families of proteins involved in adaptation and interference. CRISPR loci encode small CRISPR RNAs (crRNAs) that contain a full spacer flanked by partial repeat sequences2, 17–19. CrRNA spacers are thought to identify targets by direct Watson-Crick pairing with invasive “protospacer” DNA2, 3, but how they avoid targeting the spacer DNA within the encoding CRISPR locus itself is unknown. Here we have defined the mechanism of CRISPR self/non-self discrimination. In Staphylococcus epidermidis, target/crRNA mismatches at specific positions outside of the spacer sequence license foreign DNA for interference, whereas extended pairing between crRNA and CRISPR DNA repeats prevents autoimmunity. Hence, this CRISPR system uses the base-pairing potential of crRNAs not only to specify a target but also to spare the bacterial chromosome from interference. Differential complementarity outside of the spacer sequence is a built-in feature of all CRISPR systems, suggesting that this mechanism is a broadly applicable solution to the self/non-self dilemma that confronts all immune pathways.
doi:10.1038/nature08703
PMCID: PMC2813891  PMID: 20072129
12.  Cytotoxic Chromosomal Targeting by CRISPR/Cas Systems Can Reshape Bacterial Genomes and Expel or Remodel Pathogenicity Islands 
PLoS Genetics  2013;9(4):e1003454.
In prokaryotes, clustered regularly interspaced short palindromic repeats (CRISPRs) and their associated (Cas) proteins constitute a defence system against bacteriophages and plasmids. CRISPR/Cas systems acquire short spacer sequences from foreign genetic elements and incorporate these into their CRISPR arrays, generating a memory of past invaders. Defence is provided by short non-coding RNAs that guide Cas proteins to cleave complementary nucleic acids. While most spacers are acquired from phages and plasmids, there are examples of spacers that match genes elsewhere in the host bacterial chromosome. In Pectobacterium atrosepticum the type I-F CRISPR/Cas system has acquired a self-complementary spacer that perfectly matches a protospacer target in a horizontally acquired island (HAI2) involved in plant pathogenicity. Given the paucity of experimental data about CRISPR/Cas–mediated chromosomal targeting, we examined this process by developing a tightly controlled system. Chromosomal targeting was highly toxic via targeting of DNA and resulted in growth inhibition and cellular filamentation. The toxic phenotype was avoided by mutations in the cas operon, the CRISPR repeats, the protospacer target, and protospacer-adjacent motif (PAM) beside the target. Indeed, the natural self-targeting spacer was non-toxic due to a single nucleotide mutation adjacent to the target in the PAM sequence. Furthermore, we show that chromosomal targeting can result in large-scale genomic alterations, including the remodelling or deletion of entire pre-existing pathogenicity islands. These features can be engineered for the targeted deletion of large regions of bacterial chromosomes. In conclusion, in DNA–targeting CRISPR/Cas systems, chromosomal interference is deleterious by causing DNA damage and providing a strong selective pressure for genome alterations, which may have consequences for bacterial evolution and pathogenicity.
Author Summary
Bacteria have evolved mechanisms that provide protection from continual invasion by viruses and other foreign elements. Resistance systems, known as CRISPR/Cas, were recently discovered and equip bacteria and archaea with an “adaptive immune system.” This adaptive immunity provides a highly evolvable sequence-specific small RNA–based memory of past invasions by viruses and foreign genetic elements. There are many cases where these systems appear to target regions within the bacterial host's own genome (a possible autoimmunity), but the evolutionary rationale for this is unclear. Here, we demonstrate that CRISPR/Cas targeting of the host chromosome is highly toxic but that cells survive through mutations that alleviate the immune mechanism. We have used this phenotype to gain insight into how these systems function and show that large changes in the bacterial genome can occur. For example, targeting of a chromosomal pathogenicity island, important for virulence of the potato pathogen Pectobacterium atrosepticum, resulted in deletion of the island, which constituted ∼2% of the bacterial genome. These results have broad significance for the role of CRISPR/Cas systems and their impact on the evolution of bacterial genomes and virulence. In addition, this study demonstrates their potential as a tool for the targeted deletion of specific regions of bacterial chromosomes.
doi:10.1371/journal.pgen.1003454
PMCID: PMC3630108  PMID: 23637624
13.  Transcription, Processing, and Function of CRISPR Cassettes in Escherichia coli 
Molecular microbiology  2010;77(6):1367-1379.
CRISPR/Cas, bacterial and archaeal systems of interference with foreign genetic elements such as viruses or plasmids, consist of DNA loci called CRISPR cassettes (a set of variable spacers regularly separated by palindromic repeats) and associated cas genes. When a CRISPR spacer sequence exactly matches a sequence in a viral genome, the cell can become resistant to the virus. The CRISPR/Cas systems function through small RNAs originating from longer CRISPR cassette transcripts. While laboratory strains of Escherichia coli contain a functional CRISPR/Cas system (as judged by appearance of phage resistance at conditions of artificial co-overexpression of Cas genes and a CRISPR cassette engineered to target a λ phage), no natural phage resistance due to CRISPR system function was observed in this best-studied organism and no E. coli CRISPR spacer matches sequences of well-studied E. coli phages. To better understand the apparently “silent” E. coli CRISPR/Cas system, we systematically characterized processed transcripts from CRISPR cassettes. Using an engineered strain with genomically located spacer matching phage λ we show that endogenous levels of CRISPR cassette and cas genes expression allow only weak protection against infection with the phage. However, derepression of the CRISPR/Cas system by disruption of the hns gene leads to high level of protection.
doi:10.1111/j.1365-2958.2010.07265.x
PMCID: PMC2939963  PMID: 20624226
14.  Mobile CRISPR/Cas-Mediated Bacteriophage Resistance in Lactococcus lactis 
PLoS ONE  2012;7(12):e51663.
Lactococcus lactis is a biotechnological workhorse for food fermentations and potentially therapeutic products and is therefore widely consumed by humans. It is predominantly used as a starter microbe for fermented dairy products, and specialized strains have adapted from a plant environment through reductive evolution and horizontal gene transfer as evidenced by the association of adventitious traits with mobile elements. Specifically, L. lactis has armed itself with a myriad of plasmid-encoded bacteriophage defensive systems to protect against viral predation. This known arsenal had not included CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins), which forms a remarkable microbial immunity system against invading DNA. Although CRISPR/Cas systems are common in the genomes of closely related lactic acid bacteria (LAB), none was identified within the eight published lactococcal genomes. Furthermore, a PCR-based search of the common LAB CRISPR/Cas systems (Types I and II) in 383 industrial L. lactis strains proved unsuccessful. Here we describe a novel, Type III, self-transmissible, plasmid-encoded, phage-interfering CRISPR/Cas discovered in L. lactis. The native CRISPR spacers confer resistance based on sequence identity to corresponding lactococcal phage. The interference is directed at phages problematic to the dairy industry, indicative of a responsive system. Moreover, targeting could be modified by engineering the spacer content. The 62.8-kb plasmid was shown to be conjugally transferrable to various strains. Its mobility should facilitate dissemination within microbial communities and provide a readily applicable system to naturally introduce CRISPR/Cas to industrially relevant strains for enhanced phage resistance and prevention against acquisition of undesirable genes.
doi:10.1371/journal.pone.0051663
PMCID: PMC3519859  PMID: 23240053
15.  Structure and Genetic Content of the Megaplasmids of Neurotoxigenic Clostridium butyricum Type E Strains from Italy 
PLoS ONE  2013;8(8):e71324.
We determined the genetic maps of the megaplasmids of six neutoroxigenic Clostridium butyricum type E strains from Italy using molecular and bioinformatics techniques. The megaplasmids are circular, not linear as we had previously proposed. The differently-sized megaplasmids share a genetic region that includes structural, metabolic and regulatory genes. In addition, we found that a 168 kb genetic region is present only in the larger megaplasmids of two tested strains, whereas it is absent from the smaller megaplasmids of the four remaining strains. The genetic region unique to the larger megaplasmids contains, among other features, a locus for clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated (cas) genes, i.e. a bacterial adaptive immune system providing sequence-specific protection from invading genetic elements. Some CRISPR spacer sequences of the neurotoxigenic C. butyricum type E strains showed homology to prophage, phage and plasmid sequences from closely related clostridia species or from distant species, all sharing the intestinal habitat, suggesting that the CRISPR locus might be involved in the microorganism adaptation to the human or animal intestinal environment. Besides, we report here that each of four distinct CRISPR spacers partially matched DNA sequences of different prophages and phages, at identical nucleotide locations. This suggests that, at least in neurotoxigenic C. butyricum type E, the CRISPR locus is potentially able to recognize the same conserved DNA sequence of different invading genetic elements, besides targeting sequences unique to previously encountered invading DNA, as currently predicted for a CRISPR locus. Thus, the results of this study introduce the possibility that CRISPR loci can provide resistance to a wider range of invading DNA elements than previously appreciated. Whether it is more advantageous for the peculiar neurotoxigenic C. butyricum type E strains to maintain or to lose the CRISPR-cas system remains an open question.
doi:10.1371/journal.pone.0071324
PMCID: PMC3744543  PMID: 23967192
16.  An Archaeal Immune System Can Detect Multiple Protospacer Adjacent Motifs (PAMs) to Target Invader DNA* 
The Journal of Biological Chemistry  2012;287(40):33351-33363.
Background: CRISPR/Cas systems allow archaea and bacteria to resist invasion by foreign nucleic acids.
Results: The CRISPR/Cas system in Haloferax recognized six different PAM sequences that could trigger a defense response.
Conclusion: The PAM sequence specificity of the defense response in type I CRISPR systems is more relaxed than previously thought.
Significance: The PAM sequence requirements for interference and adaptation appear to differ markedly.
The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system provides adaptive and heritable immunity against foreign genetic elements in most archaea and many bacteria. Although this system is widespread and diverse with many subtypes, only a few species have been investigated to elucidate the precise mechanisms for the defense of viruses or plasmids. Approximately 90% of all sequenced archaea encode CRISPR/Cas systems, but their molecular details have so far only been examined in three archaeal species: Sulfolobus solfataricus, Sulfolobus islandicus, and Pyrococcus furiosus. Here, we analyzed the CRISPR/Cas system of Haloferax volcanii using a plasmid-based invader assay. Haloferax encodes a type I-B CRISPR/Cas system with eight Cas proteins and three CRISPR loci for which the identity of protospacer adjacent motifs (PAMs) was unknown until now. We identified six different PAM sequences that are required upstream of the protospacer to permit target DNA recognition. This is only the second archaeon for which PAM sequences have been determined, and the first CRISPR group with such a high number of PAM sequences. Cells could survive the plasmid challenge if their CRISPR/Cas system was altered or defective, e.g. by deletion of the cas gene cassette. Experimental PAM data were supplemented with bioinformatics data on Haloferax and Haloquadratum.
doi:10.1074/jbc.M112.377002
PMCID: PMC3460438  PMID: 22767603
Archaea; Microbiology; RNA; RNA Metabolism; RNA Processing; CRISPR/Cas; Haloferax volcanii; PAM
17.  Dealing with the Evolutionary Downside of CRISPR Immunity: Bacteria and Beneficial Plasmids 
PLoS Genetics  2013;9(9):e1003844.
The immune systems that protect organisms from infectious agents invariably have a cost for the host. In bacteria and archaea CRISPR-Cas loci can serve as adaptive immune systems that protect these microbes from infectiously transmitted DNAs. When those DNAs are borne by lytic viruses (phages), this protection can provide a considerable advantage. CRISPR-Cas immunity can also prevent cells from acquiring plasmids and free DNA bearing genes that increase their fitness. Here, we use a combination of experiments and mathematical-computer simulation models to explore this downside of CRISPR-Cas immunity and its implications for the maintenance of CRISPR-Cas loci in microbial populations. We analyzed the conjugational transfer of the staphylococcal plasmid pG0400 into Staphylococcus epidermidis RP62a recipients that bear a CRISPR-Cas locus targeting this plasmid. Contrary to what is anticipated for lytic phages, which evade CRISPR by mutations in the target region, the evasion of CRISPR immunity by plasmids occurs at the level of the host through loss of functional CRISPR-Cas immunity. The results of our experiments and models indicate that more than 10−4 of the cells in CRISPR-Cas positive populations are defective or deleted for the CRISPR-Cas region and thereby able to receive and carry the plasmid. Most intriguingly, the loss of CRISPR function even by large deletions can have little or no fitness cost in vitro. These theoretical and experimental results can account for the considerable variation in the existence, number and function of CRISPR-Cas loci within and between bacterial species. We postulate that as a consequence of the opposing positive and negative selection for immunity, CRISPR-Cas systems are in a continuous state of flux. They are lost when they bear immunity to laterally transferred beneficial genes, re-acquired by horizontal gene transfer, and ascend in environments where phage are a major source of mortality.
Author Summary
In addition to the virtue of protecting archaea and bacteria from the ravages of lethal viruses (phage), the immunity generated by the CRISPR-Cas systems have an evolutionary downside; they can prevent the acquisition of genes and genetic elements required for the adaptation and even the survival of these microbes. Using mathematical models and experiments with Staphylococcus epidermidis and the staphylococcal conjugative plasmid pG0400, we explore how bacteria deal with this evolutionary downside of CRISPR-Cas immunity. Although there are mechanisms by which immune populations of bacteria can acquire essential plasmids without the loss of CRISPR-Cas immunity, the results of our conjugation and fitness cost experiments suggest the most likely mechanism is the deactivation and deletion of this region. These results provide an explanation for the considerable variation in the existence, number and function of CRISPR-Cas within and between species of microbes. Along with other observations our work also suggests that the CRISPR-Cas loci are in a continuous state of flux: acquired by horizontal gene transfer, ascend when populations are confronted with phage and are rapidly lost when infectiously transmitted genes and genetic elements are required for the adaptation and survival of the population.
doi:10.1371/journal.pgen.1003844
PMCID: PMC3784566  PMID: 24086164
18.  CRISPR/cas Loci of Type II Propionibacterium acnes Confer Immunity against Acquisition of Mobile Elements Present in Type I P. acnes 
PLoS ONE  2012;7(3):e34171.
Propionibacterium acnes is a skin commensal that occasionally acts as an opportunistic pathogen. The population structure of this species shows three main lineages (I–III). While type I strains are mainly associated with sebaceous follicles of human skin and inflammatory acne, types II and III strains are more often associated with deep tissue infections. We investigated the occurrence and distribution of the clustered regularly interspaced short palindromic repeats (CRISPR) in P. acnes, assessed their immunological memory, and addressed the question if such a system could account for type-specific properties of the species. A collection of 108 clinical isolates covering all known phylotypes of P. acnes was screened for the existence of CRISPR/cas loci. We found that CRISPR loci are restricted to type II P. acnes strains. Sequence analyses of the CRISPR spacers revealed that the system confers immunity to P. acnes-specific phages and to two mobile genetic elements. These elements are found almost exclusively in type I P. acnes strains. Genome sequencing of a type I P. acnes isolate revealed that one element, 54 kb in size, encodes a putative secretion/tight adherence (TAD) system. Thus, CRISPR/cas loci in P. acnes recorded the exposure of type II strains to mobile genetic elements of type I strains. The CRISPR/cas locus is deleted in type I strains, which conceivably accounts for their ability to horizontally acquire fitness or virulence traits and might indicate that type I strains constitute a younger subpopulation of P. acnes.
doi:10.1371/journal.pone.0034171
PMCID: PMC3316620  PMID: 22479553
19.  Abundant and Diverse Clustered Regularly Interspaced Short Palindromic Repeat Spacers in Clostridium difficile Strains and Prophages Target Multiple Phage Types within This Pathogen 
mBio  2014;5(5):e01045-13.
ABSTRACT
Clostridium difficile is an important human-pathogenic bacterium causing antibiotic-associated nosocomial infections worldwide. Mobile genetic elements and bacteriophages have helped shape C. difficile genome evolution. In many bacteria, phage infection may be controlled by a form of bacterial immunity called the clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas) system. This uses acquired short nucleotide sequences (spacers) to target homologous sequences (protospacers) in phage genomes. C. difficile carries multiple CRISPR arrays, and in this paper we examine the relationships between the host- and phage-carried elements of the system. We detected multiple matches between spacers and regions in 31 C. difficile phage and prophage genomes. A subset of the spacers was located in prophage-carried CRISPR arrays. The CRISPR spacer profiles generated suggest that related phages would have similar host ranges. Furthermore, we show that C. difficile strains of the same ribotype could either have similar or divergent CRISPR contents. Both synonymous and nonsynonymous mutations in the protospacer sequences were identified, as well as differences in the protospacer adjacent motif (PAM), which could explain how phages escape this system. This paper illustrates how the distribution and diversity of CRISPR spacers in C. difficile, and its prophages, could modulate phage predation for this pathogen and impact upon its evolution and pathogenicity.
IMPORTANCE
Clostridium difficile is a significant bacterial human pathogen which undergoes continual genome evolution, resulting in the emergence of new virulent strains. Phages are major facilitators of genome evolution in other bacterial species, and we use sequence analysis-based approaches in order to examine whether the CRISPR/Cas system could control these interactions across divergent C. difficile strains. The presence of spacer sequences in prophages that are homologous to phage genomes raises an extra level of complexity in this predator-prey microbial system. Our results demonstrate that the impact of phage infection in this system is widespread and that the CRISPR/Cas system is likely to be an important aspect of the evolutionary dynamics in C. difficile.
doi:10.1128/mBio.01045-13
PMCID: PMC4173771  PMID: 25161187
20.  The Population and Evolutionary Dynamics of Phage and Bacteria with CRISPR–Mediated Immunity 
PLoS Genetics  2013;9(3):e1003312.
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), together with associated genes (cas), form the CRISPR–cas adaptive immune system, which can provide resistance to viruses and plasmids in bacteria and archaea. Here, we use mathematical models, population dynamic experiments, and DNA sequence analyses to investigate the host–phage interactions in a model CRISPR–cas system, Streptococcus thermophilus DGCC7710 and its virulent phage 2972. At the molecular level, the bacteriophage-immune mutant bacteria (BIMs) and CRISPR–escape mutant phage (CEMs) obtained in this study are consistent with those anticipated from an iterative model of this adaptive immune system: resistance by the addition of novel spacers and phage evasion of resistance by mutation in matching sequences or flanking motifs. While CRISPR BIMs were readily isolated and CEMs generated at high rates (frequencies in excess of 10−6), our population studies indicate that there is more to the dynamics of phage–host interactions and the establishment of a BIM–CEM arms race than predicted from existing assumptions about phage infection and CRISPR–cas immunity. Among the unanticipated observations are: (i) the invasion of phage into populations of BIMs resistant by the acquisition of one (but not two) spacers, (ii) the survival of sensitive bacteria despite the presence of high densities of phage, and (iii) the maintenance of phage-limited communities due to the failure of even two-spacer BIMs to become established in populations with wild-type bacteria and phage. We attribute (i) to incomplete resistance of single-spacer BIMs. Based on the results of additional modeling and experiments, we postulate that (ii) and (iii) can be attributed to the phage infection-associated production of enzymes or other compounds that induce phenotypic phage resistance in sensitive bacteria and kill resistant BIMs. We present evidence in support of these hypotheses and discuss the implications of these results for the ecology and (co)evolution of bacteria and phage.
Author Summary
The evidence that the CRISPR regions of the genomes of archaea and bacteria play a role in the ecology and (co)evolution of these microbes and their viruses is overwhelming: (i) the spacers (variable sequences of 26–72 bp of DNA between the repeats of this region) of these prokaryotes are homologous to the DNA of viruses in their communities; (ii) experimentally, the acquisition and incorporation of spacers of viral DNA can protect these organisms from subsequent infection by these viruses; (iii) experimentally, viruses evade this immunity by mutation in homologous protospacers or protospacer-adjacent motifs (PAMs). Not so clear are the nature and magnitude of the role CRISPR plays in this ecology and evolution. Here, we use mathematical models, experiments with Streptococcus thermophilus and the phage 2972, and DNA sequence analyses to explore the contribution of CRISPR–cas immunity to the ecology and (co)evolution of bacteria and their viruses. The results of this study suggest that the contribution of CRISPR to the ecology of bacteria and phage is more modest and limited, and the conditions for a CRISPR–mediated coevolutionary arms race between these organisms more restrictive, than anticipated from models based on the canonical view of phage infection and CRISPR–cas immunity.
doi:10.1371/journal.pgen.1003312
PMCID: PMC3597502  PMID: 23516369
21.  In defense of phage 
RNA Biology  2013;10(5):886-890.
Viruses that infect bacteria are the most abundant biological agents on the planet and bacteria have evolved diverse defense mechanisms to combat these genetic parasites. One of these bacterial defense systems relies on a repetitive locus, referred to as a CRISPR (clusters of regularly interspaced short palindromic repeats). Bacteria and archaea acquire resistance to invading viruses and plasmids by integrating short fragments of foreign nucleic acids at one end of the CRISPR locus. CRISPR loci are transcribed and the long primary CRISPR transcript is processed into a library of small RNAs that guide the immune system to invading nucleic acids, which are subsequently degraded by dedicated nucleases. However, the development of CRISPR-mediated immune systems has not eradicated phages, suggesting that viruses have evolved mechanisms to subvert CRISPR-mediated protection. Recently, Bondy-Denomy and colleagues discovered several phage-encoded anti-CRISPR proteins that offer new insight into the ongoing molecular arms race between viral parasites and the immune systems of their hosts.
doi:10.4161/rna.23591
PMCID: PMC3737345  PMID: 23392292
phage; bacterial immunity; RNA-guided immunity; anti-CRISPR; viral suppressors of RNAi (VSR); viral suppressors of CRISPR (VSC)
22.  Intricate Interactions between the Bloom-Forming Cyanobacterium Microcystis aeruginosa and Foreign Genetic Elements, Revealed by Diversified Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) Signatures 
Applied and Environmental Microbiology  2012;78(15):5353-5360.
Clustered regularly interspaced short palindromic repeats (CRISPR) confer sequence-dependent, adaptive resistance in prokaryotes against viruses and plasmids via incorporation of short sequences, called spacers, derived from foreign genetic elements. CRISPR loci are thus considered to provide records of past infections. To describe the host-parasite (i.e., cyanophages and plasmids) interactions involving the bloom-forming freshwater cyanobacterium Microcystis aeruginosa, we investigated CRISPR in four M. aeruginosa strains and in two previously sequenced genomes. The number of spacers in each locus was larger than the average among prokaryotes. All spacers were strain specific, except for a string of 11 spacers shared in two closely related strains, suggesting diversification of the loci. Using CRISPR repeat-based PCR, 24 CRISPR genotypes were identified in a natural cyanobacterial community. Among 995 unique spacers obtained, only 10 sequences showed similarity to M. aeruginosa phage Ma-LMM01. Of these, six spacers showed only silent or conservative nucleotide mutations compared to Ma-LMM01 sequences, suggesting a strategy by the cyanophage to avert CRISPR immunity dependent on nucleotide identity. These results imply that host-phage interactions can be divided into M. aeruginosa-cyanophage combinations rather than pandemics of population-wide infectious cyanophages. Spacer similarity also showed frequent exposure of M. aeruginosa to small cryptic plasmids that were observed only in a few strains. Thus, the diversification of CRISPR implies that M. aeruginosa has been challenged by diverse communities (almost entirely uncharacterized) of cyanophages and plasmids.
doi:10.1128/AEM.00626-12
PMCID: PMC3416447  PMID: 22636003
23.  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 
Biology Direct  2006;1:7.
Background
All archaeal and many bacterial genomes contain Clustered Regularly Interspaced Short Palindrome Repeats (CRISPR) and variable arrays of the CRISPR-associated (cas) genes that have been previously implicated in a novel form of DNA repair on the basis of comparative analysis of their protein product sequences. However, the proximity of CRISPR and cas genes strongly suggests that they have related functions which is hard to reconcile with the repair hypothesis.
Results
The protein sequences of the numerous cas gene products were classified into ~25 distinct protein families; several new functional and structural predictions are described. Comparative-genomic analysis of CRISPR and cas genes leads to the hypothesis that the CRISPR-Cas system (CASS) is a mechanism of defense against invading phages and plasmids that functions analogously to the eukaryotic RNA interference (RNAi) systems. Specific functional analogies are drawn between several components of CASS and proteins involved in eukaryotic RNAi, including the double-stranded RNA-specific helicase-nuclease (dicer), the endonuclease cleaving target mRNAs (slicer), and the RNA-dependent RNA polymerase. However, none of the CASS components is orthologous to its apparent eukaryotic functional counterpart. It is proposed that unique inserts of CRISPR, some of which are homologous to fragments of bacteriophage and plasmid genes, function as prokaryotic siRNAs (psiRNA), by base-pairing with the target mRNAs and promoting their degradation or translation shutdown. Specific hypothetical schemes are developed for the functioning of the predicted prokaryotic siRNA system and for the formation of new CRISPR units with unique inserts encoding psiRNA conferring immunity to the respective newly encountered phages or plasmids. The unique inserts in CRISPR show virtually no similarity even between closely related bacterial strains which suggests their rapid turnover, on evolutionary scale. Corollaries of this finding are that, even among closely related prokaryotes, the most commonly encountered phages and plasmids are different and/or that the dominant phages and plasmids turn over rapidly.
Conclusion
We proposed previously that Cas proteins comprise a novel DNA repair system. The association of the cas genes with CRISPR and, especially, the presence, in CRISPR units, of unique inserts homologous to phage and plasmid genes make us abandon this hypothesis. It appears most likely that CASS is a prokaryotic system of defense against phages and plasmids that functions via the RNAi mechanism. The functioning of this system seems to involve integration of fragments of foreign genes into archaeal and bacterial chromosomes yielding heritable immunity to the respective agents. However, it appears that this inheritance is extremely unstable on the evolutionary scale such that the repertoires of unique psiRNAs are completely replaced even in closely related prokaryotes, presumably, in response to rapidly changing repertoires of dominant phages and plasmids.
This article was reviewed by: Eric Bapteste, Patrick Forterre, and Martijn Huynen.
Open peer review
Reviewed by Eric Bapteste, Patrick Forterre, and Martijn Huynen.
For the full reviews, please go to the Reviewers' comments section.
doi:10.1186/1745-6150-1-7
PMCID: PMC1462988  PMID: 16545108
24.  CRISPR Inhibition of Prophage Acquisition in Streptococcus pyogenes 
PLoS ONE  2011;6(5):e19543.
Streptococcus pyogenes, one of the major human pathogens, is a unique species since it has acquired diverse strain-specific virulence properties mainly through the acquisition of streptococcal prophages. In addition, S. pyogenes possesses clustered regularly interspaced short palindromic repeats (CRISPR)/Cas systems that can restrict horizontal gene transfer (HGT) including phage insertion. Therefore, it was of interest to examine the relationship between CRISPR and acquisition of prophages in S. pyogenes. Although two distinct CRISPR loci were found in S. pyogenes, some strains lacked CRISPR and these strains possess significantly more prophages than CRISPR harboring strains. We also found that the number of spacers of S. pyogenes CRISPR was less than for other streptococci. The demonstrated spacer contents, however, suggested that the CRISPR appear to limit phage insertions. In addition, we found a significant inverse correlation between the number of spacers and prophages in S. pyogenes. It was therefore suggested that S. pyogenes CRISPR have permitted phage insertion by lacking its own spacers. Interestingly, in two closely related S. pyogenes strains (SSI-1 and MGAS315), CRISPR activity appeared to be impaired following the insertion of phage genomes into the repeat sequences. Detailed analysis of this prophage insertion site suggested that MGAS315 is the ancestral strain of SSI-1. As a result of analysis of 35 additional streptococcal genomes, it was suggested that the influences of the CRISPR on the phage insertion vary among species even within the same genus. Our results suggested that limitations in CRISPR content could explain the characteristic acquisition of prophages and might contribute to strain-specific pathogenesis in S. pyogenes.
doi:10.1371/journal.pone.0019543
PMCID: PMC3089615  PMID: 21573110
25.  Lactobacillus buchneri Genotyping on the Basis of Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) Locus Diversity 
Clustered regularly interspaced short palindromic repeats (CRISPR) in combination with associated sequences (cas) constitute the CRISPR-Cas immune system, which uptakes DNA from invasive genetic elements as novel “spacers” that provide a genetic record of immunization events. We investigated the potential of CRISPR-based genotyping of Lactobacillus buchneri, a species relevant for commercial silage, bioethanol, and vegetable fermentations. Upon investigating the occurrence and diversity of CRISPR-Cas systems in Lactobacillus buchneri genomes, we observed a ubiquitous occurrence of CRISPR arrays containing a 36-nucleotide (nt) type II-A CRISPR locus adjacent to four cas genes, including the universal cas1 and cas2 genes and the type II signature gene cas9. Comparative analysis of CRISPR spacer content in 26 L. buchneri pickle fermentation isolates associated with spoilage revealed 10 unique locus genotypes that contained between 9 and 29 variable spacers. We observed a set of conserved spacers at the ancestral end, reflecting a common origin, as well as leader-end polymorphisms, reflecting recent divergence. Some of these spacers showed perfect identity with phage sequences, and many spacers showed homology to Lactobacillus plasmid sequences. Following a comparative analysis of sequences immediately flanking protospacers that matched CRISPR spacers, we identified a novel putative protospacer-adjacent motif (PAM), 5′-AAAA-3′. Overall, these findings suggest that type II-A CRISPR-Cas systems are valuable for genotyping of L. buchneri.
doi:10.1128/AEM.03015-13
PMCID: PMC3911191  PMID: 24271175

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