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The CRISPR/Cas system, comprised of clustered regularly interspaced short palindromic repeats along with their associated (Cas) proteins, protects bacteria and archaea from viral predation and invading nucleic acids. While the mechanism of action for this acquired immunity is currently under investigation, the response of Cas protein expression to phage infection has yet to be elucidated. In this study, we employed shotgun proteomics to measure the global proteome expression in a model system for studying the CRISPR/Cas response in S. thermophilus DGCC7710 infected with phage 2972. Host and viral proteins were simultaneously measured following inoculation at two different multiplicities of infection and across various time points using two-dimensional liquid chromatography tandem mass spectrometry. Thirty-seven out of forty predicted viral proteins were detected, including all proteins of the structural virome and viral effector proteins. In total, 1,013 of 2,079 predicted S. thermophilus proteins were detected, facilitating the monitoring of host protein synthesis changes in response to virus infection. Importantly, Cas proteins from all four CRISPR loci in the S. thermophilus DGCC7710 genome were detected, including loci previously thought to be inactive. Many Cas proteins were found to be constitutively expressed, but several demonstrated increased abundance following infection, including the signature Cas9 proteins from the CRISPR1 and CRISPR3 loci, which are key players in the interference phase of the CRISPR/Cas response. Altogether, these results provide novel insights into the proteomic response of S. thermophilus, specifically CRISPR-associated proteins, upon phage 2972 infection. © 2012 Young et al.

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.

The categorisation and structural analysis of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) sequences from 195 microbial genomes show that repeats from diverse organisms can be grouped based on sequence similarity, and that some groups have pronounced secondary structures with compensatory base changes., Background Clustered regularly interspaced short palindromic repeats (CRISPRs) are a novel class of direct repeats, separated by unique spacer sequences of similar length, that are present in approximately 40% of bacterial and most archaeal genomes analyzed to date. More than 40 gene families, called CRISPR-associated sequences (CASs), appear in conjunction with these repeats and are thought to be involved in the propagation and functioning of CRISPRs. It has been recently shown that CRISPR provides acquired resistance against viruses in prokaryotes. Results Here we analyze CRISPR repeats identified in 195 microbial genomes and show that they can be organized into multiple clusters based on sequence similarity. Some of the clusters present stable, highly conserved RNA secondary structures, while others lack detectable structures. Stable secondary structures exhibit multiple compensatory base changes in the stem region, indicating evolutionary and functional conservation. Conclusion We show that the repeat-based classification corresponds to, and expands upon, a previously reported CAS gene-based classification, including specific relationships between CRISPR and CAS subtypes.

SummaryIn the face of infectious disease, organisms evolved a range of defense mechanisms, with a clear distinction between those that are constitutive (always active) and those that are inducible (elicited by parasites) [1]. Both defense strategies have evolved from each other [2], but we lack an understanding of the conditions that favor one strategy over the other. While it is hard to generalize about their degree of protection, it is possible to make generalizations about their associated fitness costs, which are commonly detected [3–5]. By definition, constitutive defenses are always “on,” and are therefore associated with a fixed cost, independent of parasite exposure [4, 5]. Inducible defenses, on the other hand, may lack costs in the absence of parasites but become costly when defense is elicited [6] through processes such as immunopathology [7]. Bacteria can evolve constitutive defense against phage by modification/masking of surface receptors [8, 9], which is often associated with reduced fitness in the absence of phage [10]. Bacteria can also evolve inducible defense using the CRISPR-Cas (clustered regularly interspaced short palindromic repeat, CRISPR associated) immune system [11], which is typically elicited upon infection [12–14]. CRISPR-Cas functions by integrating phage sequences into CRISPR loci on the host genome [15]. Upon re-infection, CRISPR transcripts guide cleavage of phage genomes [16–20]. In nature, both mechanisms are important [21, 22]. Using a general theoretical model and experimental evolution, we tease apart the mechanism that drives their evolution and show that infection risk determines the relative investment in the two arms of defense.

About half of all bacteria carry genes for CRISPR-Cas adaptive immune systems1, which provide immunological memory by inserting short DNA sequences from phage and other parasitic DNA elements into CRISPR loci on the host genome2. Whereas CRISPR loci evolve rapidly in natural environments3,4, bacterial species typically evolve phage resistance by the mutation or loss of phage receptors under laboratory conditions5,6. Here we report how this discrepancy may in part be explained by differences in the biotic complexity of in vitro and natural environments7,8. Specifically, by using the opportunistic pathogen Pseudomonas aeruginosa and its phage DMS3vir, we show that coexistence with other human pathogens amplifies the fitness trade-offs associated with the mutation of phage receptors, and therefore tips the balance in favour of the evolution of CRISPR-based resistance. We also demonstrate that this has important knock-on effects for the virulence of P. aeruginosa, which became attenuated only if the bacteria evolved surface-based resistance. Our data reveal that the biotic complexity of microbial communities in natural environments is an important driver of the evolution of CRISPR-Cas adaptive immunity, with key implications for bacterial fitness and virulence.

SummaryCompelling evidence indicates that the CRISPR-Cas system protects prokaryotes from viruses and other potential genome invaders. This adaptive prokaryotic immune system arises from the clustered regularly interspaced short palindromic repeats (CRISPRs) found in prokaryotic genomes, which harbor short invader-derived sequences, and the CRISPR-associated (Cas) protein-coding genes. Here, we have identified a CRISPR-Cas effector complex that is comprised of small invader-targeting RNAs from the CRISPR loci (termed prokaryotic silencing (psi)RNAs) and the RAMP module (or Cmr) Cas proteins. The psiRNA-Cmr protein complexes cleave complementary target RNAs at a fixed distance from the 3′ end of the integral psiRNAs. In Pyrococcus furiosus, psiRNAs occur in two size forms that share a common 5′ sequence tag but have distinct 3′ ends that direct cleavage of a given target RNA at two distinct sites. Our results indicate that prokaryotes possess a unique RNA silencing system that functions by homology-dependent cleavage of invader RNAs.

CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated) systems allow bacteria to adapt to infection by acquiring 'spacer' sequences from invader DNA into genomic CRISPR loci. Cas proteins use RNAs derived from these loci to target cognate sequences for destruction through CRISPR interference. Mutations in the protospacer adjacent motif (PAM) and seed regions block interference but promote rapid 'primed' adaptation. Here, we use multiple spacer sequences to reexamine the PAM and seed sequence requirements for interference and priming in the Escherichia coli Type I-E CRISPR-Cas system. Surprisingly, CRISPR interference is far more tolerant of mutations in the seed and the PAM than previously reported, and this mutational tolerance, as well as priming activity, is highly dependent on spacer sequence. We identify a large number of functional PAMs that can promote interference, priming or both activities, depending on the associated spacer sequence. Functional PAMs are preferentially acquired during unprimed 'naive' adaptation, leading to a rapid priming response following infection. Our results provide numerous insights into the importance of both spacer and target sequences for interference and priming, and reveal that priming is a major pathway for adaptation during initial infection.

CRISPR-Cas is an adaptive prokaryotic immune system that prevents phage infection. By incorporating phage-derived ‘spacer’ sequences into CRISPR loci on the host genome, future infections from the same phage genotype can be recognized and the phage genome cleaved. However, the phage can escape CRISPR degradation by mutating the sequence targeted by the spacer, allowing them to re-infect previously CRISPR-immune hosts, and theoretically leading to coevolution. Previous studies have shown that phage can persist over long periods in populations of Streptococcus thermophilus that can acquire CRISPR-Cas immunity, but it has remained less clear whether this coexistence was owing to coevolution, and if so, what type of coevolutionary dynamics were involved. In this study, we performed highly replicated serial transfer experiments over 30 days with S. thermophilus and a lytic phage. Using a combination of phenotypic and genotypic data, we show that CRISPR-mediated resistance and phage infectivity coevolved over time following an arms race dynamic, and that asymmetry between phage infectivity and host resistance within this system eventually causes phage extinction. This work provides further insight into the way CRISPR-Cas systems shape the population and coevolutionary dynamics of bacteria–phage interactions. This article is part of a discussion meeting issue ‘The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems’.

CRISPR–Cas systems are adaptive immune systems in prokaryotes, providing resistance against invading viruses and plasmids. The identification of CRISPR loci is currently a non-standardized, ambiguous process, requiring the manual combination of multiple tools, where existing tools detect only parts of the CRISPR-systems, and lack quality control, annotation and assessment capabilities of the detected CRISPR loci. Our CRISPRloci server provides the first resource for the prediction and assessment of all possible CRISPR loci. The server integrates a series of advanced Machine Learning tools within a seamless web interface featuring: (i) prediction of all CRISPR arrays in the correct orientation; (ii) definition of CRISPR leaders for each locus; and (iii) annotation of cas genes and their unambiguous classification. As a result, CRISPRloci is able to accurately determine the CRISPR array and associated information, such as: the Cas subtypes; cassette boundaries; accuracy of the repeat structure, orientation and leader sequence; virus-host interactions; self-targeting; as well as the annotation of cas genes, all of which have been missing from existing tools. This annotation is presented in an interactive interface, making it easy for scientists to gain an overview of the CRISPR system in their organism of interest. Predictions are also rendered in GFF format, enabling in-depth genome browser inspection. In summary, CRISPRloci constitutes a full suite for CRISPR–Cas system characterization that offers annotation quality previously available only after manual inspection.

Classified as the genospecies Clostridium novyi sensu lato and distributed into four lineages (I–IV), Clostridium botulinum (group III), Clostridium novyi, and Clostridium haemolyticum are clostridial pathogens that cause animal diseases. Clostridium novyi sensu lato contains a large mobilome consisting of plasmids and circular bacteriophages. Here, we explored clustered regularly interspaced short palindromic repeats (CRISPR) arrays and their associated proteins (Cas) to shed light on the link between evolution of CRISPR-Cas systems and the plasmid and phage composition in a study of 58 Clostridium novyi sensu lato genomes. In 55 of these genomes, types I-B (complete or partial), I-D, II-C, III-B, III-D, or V-U CRISPR-Cas systems were detected in chromosomes as well as in mobile genetic elements (MGEs). Type I-B predominated (67.2%) and was the only CRISPR type detected in the Ia, III, and IV genomic lineages. Putative type V-U CRISPR Cas14a genes were detected in two different cases: next to partial type-IB CRISPR loci on the phage encoding the botulinum neurotoxin (BoNT) in lineage Ia and in 12 lineage II genomes, as part of a putative integrative element related to a phage-inducible chromosomal island (PICI). In the putative PICI, Cas14a was associated with CRISPR arrays and restriction modification (RM) systems as part of an accessory locus. This is the first time a PICI containing such locus has been detected in C. botulinum. Mobilome composition and dynamics were also investigated based on the contents of the CRISPR arrays and the study of spacers. A large proportion of identified protospacers (20.2%) originated from Clostridium novyi sensu lato (p1_Cst, p4_BKT015925, p6_Cst, CWou-2020a, p1_BKT015925, and p2_BKT015925), confirming active exchanges within this genospecies and the key importance of specific MGEs in Clostridium novyi sensu lato.

Background Xanthomonads are an important clade of Gram-negative bacteria infecting a plethora of economically important host plants, including citrus. Knowledge about the pathogen’s diversity and population structure are prerequisite for epidemiological surveillance and efficient disease management. Rapidly evolving genetic loci, such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), are of special interest to develop new molecular typing tools. Results We analyzed CRISPR loci of 56 Xanthomonas citri pv. citri strains of world-wide origin, a regulated pathogen causing Asiatic citrus canker in several regions of the world. With one exception, 23 unique sequences built up the repertoire of spacers, suggesting that this set of strains originated from a common ancestor that already harbored these 23 spacers. One isolate originating from Pakistan contained a string of 14 additional, probably more recently acquired spacers indicating that this genetic lineage has or had until recently the capacity to acquire new spacers. Comparison of CRISPR arrays with previously obtained molecular typing data, such as amplified fragment length polymorphisms (AFLP), variable-number of tandem-repeats (VNTR) and genome-wide single-nucleotide polymorphisms (SNP), demonstrated that these methods reveal similar evolutionary trajectories. Notably, genome analyses allowed to generate a model for CRISPR array evolution in X. citri pv. citri, which provides a new framework for the genealogy of the citrus canker pathogen. Conclusions CRISPR-based typing will further improve the accuracy of the genetic identification of X. citri pv. citri outbreak strains in molecular epidemiology analyses, especially when used concomitantly with another genotyping method.

Mycobacterium tuberculosis complex (MTC) CRISPR locus diversity has long been studied solely investigating the presence/absence of a known set of spacers. Unveiling the genetic mechanisms of its evolution requires a more exhaustive reconstruction in a large amount of representative strains. In this article, we point out and resolve, with a new pipeline, the problem of CRISPR reconstruction based directly on short read sequences in M. tuberculosis. We first show that the process we set up, that we coin as “CRISPRbuilder-TB” (https://github.com/cguyeux/CRISPRbuilder-TB), allows an efficient reconstruction of simulated or real CRISPRs, even when including complex evolutionary steps like the insertions of mobile elements. Compared to more generalist tools, the whole process is much more precise and robust, and requires only minimal manual investigation. Second, we show that more than 1/3 of the currently complete genomes available for this complex in the public databases contain largely erroneous CRISPR loci. Third, we highlight how both the classical experimental in vitro approach and the basic in silico spoligotyping provided by existing analytic tools miss a whole diversity of this locus in MTC, by not capturing duplications, spacer and direct repeats variants, and IS6110 insertion locations. This description is extended in a second article that describes MTC-CRISPR diversity and suggests general rules for its evolution. This work opens perspectives for an in-depth exploration of M. tuberculosis CRISPR loci diversity and of mechanisms involved in its evolution and its functionality, as well as its adaptation to other CRISPR locus-harboring bacterial species.

Cascade complexes underpin E. coli CRISPR-Cas immunity systems by stimulating "adaptation" reactions that update immunity and by initiating "interference" reactions that destroy invader DNA. Recognition of invader DNA in Cascade catalysed R-loops provokes DNA capture and its subsequent integration into CRISPR loci by Cas1 and Cas2. DNA capture processes are unclear but may involve RecG helicase, which stimulates adaptation during its role responding to genome instability. We show that Cascade is a potential source of genome instability because it blocks DNA replication and that RecG helicase alleviates this by dissociating Cascade. This highlights how integrating in vitro CRISPR-Cas interference and adaptation reactions with DNA replication and repair reactions will help to determine precise mechanisms underpinning prokaryotic adaptive immunity.

The CRISPR-Cas (Clustered Regularly Interspaced Short Palindrome Repeats – CRISPR associated proteins) system provides adaptive immunity in archaea and bacteria. A hallmark of CRISPR-Cas is the involvement of short crRNAs that guide associated proteins in the destruction of invading DNA or RNA. We present three fundamentally distinct processing pathways in the cyanobacterium Synechocystis sp. PCC6803 for a subtype I-D (CRISPR1), and two type III systems (CRISPR2 and CRISPR3), which are located together on the plasmid pSYSA. Using high-throughput transcriptome analyses and assays of transcript accumulation we found all CRISPR loci to be highly expressed, but the individual crRNAs had profoundly varying abundances despite single transcription start sites for each array. In a computational analysis, CRISPR3 spacers with stable secondary structures displayed a greater ratio of degradation products. These structures might interfere with the loading of the crRNAs into RNP complexes, explaining the varying abundancies. The maturation of CRISPR1 and CRISPR2 transcripts depends on at least two different Cas6 proteins. Mutation of gene sll7090, encoding a Cmr2 protein led to the disappearance of all CRISPR3-derived crRNAs, providing in vivo evidence for a function of Cmr2 in the maturation, regulation of expression, Cmr complex formation or stabilization of CRISPR3 transcripts. Finally, we optimized CRISPR repeat structure prediction and the results indicate that the spacer context can influence individual repeat structures.

CRISPR-Cas systems are adaptive prokaryotic immune systems protecting against horizontally transferred DNA or RNA such as viruses and other mobile genetic elements. Memory of past invaders is stored as spacers in CRISPR loci in a process called adaptation. Here we developed a novel assay where spacer integration results in fluorescence, enabling detection of memory formation in single cells and quantification of as few as 0.05% cells with expanded CRISPR arrays in a bacterial population. Using this fluorescent CRISPR Adaptation Reporter (f-CAR), we quantified adaptation of the two CRISPR arrays of the type I-E CRISPR-Cas system in Escherichia coli, and confirmed that more integration events are targeted to CRISPR-II than to CRISPR-I. The f-CAR conveniently analyzes and compares many samples, allowing new insights into adaptation. For instance, we show that in an E. coli culture the majority of acquisition events occur in late exponential phase.

Clustered regularly interspaced short palindromic repeats (CRISPRs) are repetitive genetic elements found in archaeal and bacterial genomes that are involved in RNA-mediated adaptive immunity against foreign invading nucleic acids such as phages and plasmids. CRISPR-associated (cas) genes are also found adjacent to CRISPR loci and encode Cas proteins with a variety of nucleic acid-related functions. Cas5d proteins are subtype I-C specific Cas5 proteins that constitute one of the most plevalent Cas protein families in CRISPR-Cas systems, and have been predicted to have RNA-recognition motif (RRM) domains. Here, we report crystal structures of Cas5d proteins from Streptococcus pyogenes and Xanthomonas oryzae, which represent two Cas5d subgroups. S. pyogenes Cas5d contains extra C-terminal residues while X.oryzae Cas5d has an extended helical region protruding from the N-terminal RRM domain, which has not been observed in other Cas5d structures previously. Despite structural differences, the two Cas5d proteins share common functional properties such as specific endoribonuclease activity for pre-crRNA and non-specific double-stranded DNA binding. These findings suggest that Cas5d may have multiple roles in CRISPR-mediated immunity system and provide an explanation for the conserved mode of the pre-CRISPR RNA processing.

Bacteria and archaea have evolved sophisticated adaptive immune systems that reply on CRISPR loci and a diverse cassette of Cas genes that are classified into three main types and at least eleven subtypes. All CRISPR-Cas immune systems operate through three main stages: acquisition, biogenesis, and interference. This SnapShot summarizes our current knowledge of these fascinating immune systems. To view this SnapShot, open or download the PDF.

Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (cas) proteins provide RNA-mediated adaptive immunity against foreign invading nucleic acids such as phages and plasmids in archaea and bacteria. Cas2 protein is one of the two core Cas proteins are present in all types of CRISPR-Cas systems and is required for new spacer integration into CRISPR loci. Cas2 homologues from several CRISPR-Cas subtypes were characterized as metal-dependent nucleases with different substrate preferences, and it was proposed that a pH-dependent conformational change mediates metal binding and catalysis. Here, we report the X-ray crystal structures of Streptococcus pyogenes Cas2 protein at three different pHs (5.6, 6.5, and 7.5), and the results of its nuclease activity assay at varying pHs (6.0-9.0). While S. pyogenes Cas2 exhibited strongly pH-dependent catalytic activity, there was no significant conformational difference among the three crystal structures. However, structural comparisons with other Cas2 homologues suggest structural variability and the flexible nature of its putative hinge regions, supporting the supposition that conformational change is important for catalysis.

Background CRISPR (Clustered, Regularly, Interspaced, Short, Palindromic Repeats) loci provide prokaryotes with an adaptive immunity against viruses and other mobile genetic elements. CRISPR arrays can be transcribed and processed into small crRNA molecules, which are then used by the cell to target the foreign nucleic acid. Since spacers are accumulated by active CRISPR/Cas systems, the sequences of these spacers provide a record of the past "infection history" of the organism. Results Here we analyzed all currently known spacers present in archaeal genomes and identified their source by DNA similarity. While nearly 50% of archaeal spacers matched mobile genetic elements, such as plasmids or viruses, several others matched chromosomal genes of other organisms, primarily other archaea. Thus, networks of gene exchange between archaeal species were revealed by the spacer analysis, including many cases of inter-genus and inter-species gene transfer events. Spacers that recognize viral sequences tend to be located further away from the leader sequence, implying that there exists a selective pressure for their retention. Conclusions CRISPR spacers provide direct evidence for extensive gene exchange in archaea, especially within genera, and support the current dogma where the primary role of the CRISPR/Cas system is anti-viral and anti-plasmid defense. Open peer review This article was reviewed by: Profs. W. Ford Doolittle, John van der Oost, Christa Schleper (nominated by board member Prof. J Peter Gogarten)