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1.  CRISPR-spacer integration reporter plasmids reveal distinct genuine acquisition specificities among CRISPR-Cas I-E variants of Escherichia coli 
RNA Biology  2013;10(5):792-802.
Prokaryotes immunize themselves against transmissible genetic elements by the integration (acquisition) in clustered regularly interspaced short palindromic repeats (CRISPR) loci of spacers homologous to invader nucleic acids, defined as protospacers. Following acquisition, mono-spacer CRISPR RNAs (termed crRNAs) guide CRISPR-associated (Cas) proteins to degrade (interference) protospacers flanked by an adjacent motif in extrachomosomal DNA. During acquisition, selection of spacer-precursors adjoining the protospacer motif and proper orientation of the integrated fragment with respect to the leader (sequence leading transcription of the flanking CRISPR array) grant efficient interference by at least some CRISPR-Cas systems. This adaptive stage of the CRISPR action is poorly characterized, mainly due to the lack of appropriate genetic strategies to address its study and, at least in Escherichia coli, the need of Cas overproduction for insertion detection. In this work, we describe the development and application in Escherichia coli strains of an interference-independent assay based on engineered selectable CRISPR-spacer integration reporter plasmids. By using this tool without the constraint of interference or cas overexpression, we confirmed fundamental aspects of this process such as the critical requirement of Cas1 and Cas2 and the identity of the CTT protospacer motif for the E. coli K12 system. In addition, we defined the CWT motif for a non-K12 CRISPR-Cas variant, and obtained data supporting the implication of the leader in spacer orientation, the preferred acquisition from plasmids harboring cas genes and the occurrence of a sequential cleavage at the insertion site by a ruler mechanism.
doi:10.4161/rna.24023
PMCID: PMC3737337  PMID: 23445770
CRISPR-spacer acquisition; Cascade; Escherichia coli K12; O157:H7; RNA-guided immunity; cas genes; protospacer adjacent motif; reporter plasmids; ruler mechanism; spacer orientation
2.  Type I-E CRISPR-Cas Systems Discriminate Target from Non-Target DNA through Base Pairing-Independent PAM Recognition 
PLoS Genetics  2013;9(9):e1003742.
Discriminating self and non-self is a universal requirement of immune systems. Adaptive immune systems in prokaryotes are centered around repetitive loci called CRISPRs (clustered regularly interspaced short palindromic repeat), into which invader DNA fragments are incorporated. CRISPR transcripts are processed into small RNAs that guide CRISPR-associated (Cas) proteins to invading nucleic acids by complementary base pairing. However, to avoid autoimmunity it is essential that these RNA-guides exclusively target invading DNA and not complementary DNA sequences (i.e., self-sequences) located in the host's own CRISPR locus. Previous work on the Type III-A CRISPR system from Staphylococcus epidermidis has demonstrated that a portion of the CRISPR RNA-guide sequence is involved in self versus non-self discrimination. This self-avoidance mechanism relies on sensing base pairing between the RNA-guide and sequences flanking the target DNA. To determine if the RNA-guide participates in self versus non-self discrimination in the Type I-E system from Escherichia coli we altered base pairing potential between the RNA-guide and the flanks of DNA targets. Here we demonstrate that Type I-E systems discriminate self from non-self through a base pairing-independent mechanism that strictly relies on the recognition of four unchangeable PAM sequences. In addition, this work reveals that the first base pair between the guide RNA and the PAM nucleotide immediately flanking the target sequence can be disrupted without affecting the interference phenotype. Remarkably, this indicates that base pairing at this position is not involved in foreign DNA recognition. Results in this paper reveal that the Type I-E mechanism of avoiding self sequences and preventing autoimmunity is fundamentally different from that employed by Type III-A systems. We propose the exclusive targeting of PAM-flanked sequences to be termed a target versus non-target discrimination mechanism.
Author Summary
CRISPR loci and their associated genes form a diverse set of adaptive immune systems that are widespread among prokaryotes. In these systems, the CRISPR-associated genes (cas) encode for proteins that capture fragments of invading DNA and integrate these sequences between repeat sequences of the host's CRISPR locus. This information is used upon re-infection to degrade invader genomes. Storing invader sequences in host genomes necessitates a mechanism to differentiate between invader sequences on invader genomes and invader sequences on the host genome. CRISPR-Cas of Staphylococcus epidermidis (Type III-A system) is inhibited when invader sequences are flanked by repeat sequences, and this prevents targeting of the CRISPR locus on the host genome. Here we demonstrate that Escherichia coli CRISPR-Cas (Type I-E system) is not inhibited by repeat sequences. Instead, this system is specifically activated by the presence of bona fide Protospacer Adjacent Motifs (PAMs) in the target. PAMs are conserved sequences adjoining invader sequences on the invader genome, and these sequences are never adjacent to invader sequences within host CRISPR loci. PAM recognition is not affected by base pairing potential of the target with the crRNA. As such, the Type I-E system lacks the ability to specifically recognize self DNA.
doi:10.1371/journal.pgen.1003742
PMCID: PMC3764190  PMID: 24039596
3.  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
4.  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
5.  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
6.  Detection and characterization of spacer integration intermediates in type I-E CRISPR–Cas system 
Nucleic Acids Research  2014;42(12):7884-7893.
The adaptation against foreign nucleic acids by the CRISPR–Cas system (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated proteins) depends on the insertion of foreign nucleic acid-derived sequences into the CRISPR array as novel spacers by still unknown mechanism. We identified and characterized in Escherichia coli intermediate states of spacer integration and mapped the integration site at the chromosomal CRISPR array in vivo. The results show that the insertion of new spacers occurs by site-specific nicking at both strands of the leader proximal repeat in a staggered way and is accompanied by joining of the resulting 5′-ends of the repeat strands with the 3′-ends of the incoming spacer. This concerted cleavage-ligation reaction depends on the metal-binding center of Cas1 protein and requires the presence of Cas2. By acquisition assays using plasmid-located CRISPR array with mutated repeat sequences, we demonstrate that the primary sequence of the first repeat is crucial for cleavage of the CRISPR array and the ligation of new spacer DNA.
doi:10.1093/nar/gku510
PMCID: PMC4081107  PMID: 24920831
7.  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
8.  CRISPR Interference Directs Strand Specific Spacer Acquisition 
PLoS ONE  2012;7(4):e35888.
Background
CRISPR/Cas is a widespread adaptive immune system in prokaryotes. This system integrates short stretches of DNA derived from invading nucleic acids into genomic CRISPR loci, which function as memory of previously encountered invaders. In Escherichia coli, transcripts of these loci are cleaved into small RNAs and utilized by the Cascade complex to bind invader DNA, which is then likely degraded by Cas3 during CRISPR interference.
Results
We describe how a CRISPR-activated E. coli K12 is cured from a high copy number plasmid under non-selective conditions in a CRISPR-mediated way. Cured clones integrated at least one up to five anti-plasmid spacers in genomic CRISPR loci. New spacers are integrated directly downstream of the leader sequence. The spacers are non-randomly selected to target protospacers with an AAG protospacer adjacent motif, which is located directly upstream of the protospacer. A co-occurrence of PAM deviations and CRISPR repeat mutations was observed, indicating that one nucleotide from the PAM is incorporated as the last nucleotide of the repeat during integration of a new spacer. When multiple spacers were integrated in a single clone, all spacer targeted the same strand of the plasmid, implying that CRISPR interference caused by the first integrated spacer directs subsequent spacer acquisition events in a strand specific manner.
Conclusions
The E. coli Type I-E CRISPR/Cas system provides resistance against bacteriophage infection, but also enables removal of residing plasmids. We established that there is a positive feedback loop between active spacers in a cluster – in our case the first acquired spacer - and spacers acquired thereafter, possibly through the use of specific DNA degradation products of the CRISPR interference machinery by the CRISPR adaptation machinery. This loop enables a rapid expansion of the spacer repertoire against an actively present DNA element that is already targeted, amplifying the CRISPR interference effect.
doi:10.1371/journal.pone.0035888
PMCID: PMC3338789  PMID: 22558257
9.  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
10.  Priming in the Type I-F CRISPR-Cas system triggers strand-independent spacer acquisition, bi-directionally from the primed protospacer 
Nucleic Acids Research  2014;42(13):8516-8526.
Clustered regularly interspaced short palindromic repeats (CRISPR), in combination with CRISPR associated (cas) genes, constitute CRISPR-Cas bacterial adaptive immune systems. To generate immunity, these systems acquire short sequences of nucleic acids from foreign invaders and incorporate these into their CRISPR arrays as spacers. This adaptation process is the least characterized step in CRISPR-Cas immunity. Here, we used Pectobacterium atrosepticum to investigate adaptation in Type I-F CRISPR-Cas systems. Pre-existing spacers that matched plasmids stimulated hyperactive primed acquisition and resulted in the incorporation of up to nine new spacers across all three native CRISPR arrays. Endogenous expression of the cas genes was sufficient, yet required, for priming. The new spacers inhibited conjugation and transformation, and interference was enhanced with increasing numbers of new spacers. We analyzed ∼350 new spacers acquired in priming events and identified a 5′-protospacer-GG-3′ protospacer adjacent motif. In contrast to priming in Type I-E systems, new spacers matched either plasmid strand and a biased distribution, including clustering near the primed protospacer, suggested a bi-directional translocation model for the Cas1:Cas2–3 adaptation machinery. Taken together these results indicate priming adaptation occurs in different CRISPR-Cas systems, that it can be highly active in wild-type strains and that the underlying mechanisms vary.
doi:10.1093/nar/gku527
PMCID: PMC4117759  PMID: 24990370
11.  Substrate Generation for Endonucleases of CRISPR/Cas Systems 
The interaction of viruses and their prokaryotic hosts shaped the evolution of bacterial and archaeal life. Prokaryotes developed several strategies to evade viral attacks that include restriction modification, abortive infection and CRISPR/Cas systems. These adaptive immune systems found in many Bacteria and most Archaea consist of clustered regularly interspaced short palindromic repeat (CRISPR) sequences and a number of CRISPR associated (Cas) genes (Fig. 1)1-3. Different sets of Cas proteins and repeats define at least three major divergent types of CRISPR/Cas systems 4. The universal proteins Cas1 and Cas2 are proposed to be involved in the uptake of viral DNA that will generate a new spacer element between two repeats at the 5' terminus of an extending CRISPR cluster 5. The entire cluster is transcribed into a precursor-crRNA containing all spacer and repeat sequences and is subsequently processed by an enzyme of the diverse Cas6 family into smaller crRNAs 6-8. These crRNAs consist of the spacer sequence flanked by a 5' terminal (8 nucleotides) and a 3' terminal tag derived from the repeat sequence 9. A repeated infection of the virus can now be blocked as the new crRNA will be directed by a Cas protein complex (Cascade) to the viral DNA and identify it as such via base complementarity10. Finally, for CRISPR/Cas type 1 systems, the nuclease Cas3 will destroy the detected invader DNA 11,12 .
These processes define CRISPR/Cas as an adaptive immune system of prokaryotes and opened a fascinating research field for the study of the involved Cas proteins. The function of many Cas proteins is still elusive and the causes for the apparent diversity of the CRISPR/Cas systems remain to be illuminated. Potential activities of most Cas proteins were predicted via detailed computational analyses. A major fraction of Cas proteins are either shown or proposed to function as endonucleases 4.
Here, we present methods to generate crRNAs and precursor-cRNAs for the study of Cas endoribonucleases. Different endonuclease assays require either short repeat sequences that can directly be synthesized as RNA oligonucleotides or longer crRNA and pre-crRNA sequences that are generated via in vitro T7 RNA polymerase run-off transcription. This methodology allows the incorporation of radioactive nucleotides for the generation of internally labeled endonuclease substrates and the creation of synthetic or mutant crRNAs. Cas6 endonuclease activity is utilized to mature pre-crRNAs into crRNAs with 5'-hydroxyl and a 2',3'-cyclic phosphate termini.
doi:10.3791/4277
PMCID: PMC3490271  PMID: 22986408
Molecular biology; Issue 67; CRISPR/Cas; endonuclease;  in vitro transcription; crRNA; Cas6
12.  CRISPR loci reveal networks of gene exchange in archaea 
Biology Direct  2011;6:65.
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)
doi:10.1186/1745-6150-6-65
PMCID: PMC3285040  PMID: 22188759
CRISPR; Lateral Gene transfer; Horizontal gene transfer; viruses; archaea; competence
13.  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
14.  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
15.  Selective and hyperactive uptake of foreign DNA by adaptive immune systems of an archaeon via two distinct mechanisms 
Molecular Microbiology  2012;85(6):1044-1056.
Central to the disparate adaptive immune systems of archaea and bacteria are clustered regularly interspaced short palindromic repeats (CRISPR). The spacer regions derive from invading genetic elements and, via RNA intermediates and associated proteins, target and cleave nucleic acids of the invader. Here we demonstrate the hyperactive uptake of hundreds of unique spacers within CRISPR loci associated with type I and IIIB immune systems of a hyperthermophilic archaeon. Infection with an environmental virus mixture resulted in the exclusive uptake of protospacers from a co-infecting putative conjugative plasmid. Spacer uptake occurred by two distinct mechanisms in only one of two CRISPR loci subfamilies present. In two loci, insertions, often multiple, occurred adjacent to the leader while in a third locus single spacers were incorporated throughout the array. Protospacer DNAs were excised from the invading genetic element immediately after CCN motifs, on either strand, with the secondary cut apparently produced by a ruler mechanism. Over a 10-week period, there was a gradual decrease in the number of wild-type cells present in the culture but the virus and putative conjugative plasmid were still propagating. The results underline the complex dynamics of CRISPR-based immune systems within a population infected with genetic elements.
doi:10.1111/j.1365-2958.2012.08171.x
PMCID: PMC3468723  PMID: 22834906
16.  Chromosomal targeting by CRISPR-Cas systems can contribute to genome plasticity in bacteria 
Mobile Genetic Elements  2013;3(5):e26831.
The clustered regularly interspaced short palindromic repeats (CRISPR) and their associated (Cas) proteins form adaptive immune systems in bacteria to combat phage and other foreign genetic elements. Typically, short spacer sequences are acquired from the invader DNA and incorporated into CRISPR arrays in the bacterial genome. Small RNAs are generated that contain these spacer sequences and enable sequence-specific destruction of the foreign nucleic acids. Occasionally, spacers are acquired from the chromosome, which instead leads to targeting of the host genome. Chromosomal targeting is highly toxic to the bacterium, providing a strong selective pressure for a variety of evolutionary routes that enable host cell survival. Mutations that inactivate the CRISPR-Cas functionality, such as within the cas genes, CRISPR repeat, protospacer adjacent motifs (PAM), and target sequence, mediate escape from toxicity. This self-targeting might provide some explanation for the incomplete distribution of CRISPR-Cas systems in less than half of sequenced bacterial genomes. More importantly, self-genome targeting can cause large-scale genomic alterations, including remodeling or deletion of pathogenicity islands and other non-mobile chromosomal regions. While control of horizontal gene transfer is perceived as their main function, our recent work illuminates an alternative role of CRISPR-Cas systems in causing host genomic changes and influencing bacterial evolution.
doi:10.4161/mge.26831
PMCID: PMC3827097  PMID: 24251073
CRISPR; Cas; chromosomal targeting; bacterial evolution; genomic islands; plasmids; horizontal gene transfer; bacteriophages; integrative and conjugative elements
17.  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
18.  CRISPR-Cas Systems in the Cyanobacterium Synechocystis sp. PCC6803 Exhibit Distinct Processing Pathways Involving at Least Two Cas6 and a Cmr2 Protein 
PLoS ONE  2013;8(2):e56470.
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.
doi:10.1371/journal.pone.0056470
PMCID: PMC3575380  PMID: 23441196
19.  CRISPRstrand: predicting repeat orientations to determine the crRNA-encoding strand at CRISPR loci 
Bioinformatics  2014;30(17):i489-i496.
Motivation: The discovery of CRISPR-Cas systems almost 20 years ago rapidly changed our perception of the bacterial and archaeal immune systems. CRISPR loci consist of several repetitive DNA sequences called repeats, inter-spaced by stretches of variable length sequences called spacers. This CRISPR array is transcribed and processed into multiple mature RNA species (crRNAs). A single crRNA is integrated into an interference complex, together with CRISPR-associated (Cas) proteins, to bind and degrade invading nucleic acids. Although existing bioinformatics tools can recognize CRISPR loci by their characteristic repeat-spacer architecture, they generally output CRISPR arrays of ambiguous orientation and thus do not determine the strand from which crRNAs are processed. Knowledge of the correct orientation is crucial for many tasks, including the classification of CRISPR conservation, the detection of leader regions, the identification of target sites (protospacers) on invading genetic elements and the characterization of protospacer-adjacent motifs.
Results: We present a fast and accurate tool to determine the crRNA-encoding strand at CRISPR loci by predicting the correct orientation of repeats based on an advanced machine learning approach. Both the repeat sequence and mutation information were encoded and processed by an efficient graph kernel to learn higher-order correlations. The model was trained and tested on curated data comprising >4500 CRISPRs and yielded a remarkable performance of 0.95 AUC ROC (area under the curve of the receiver operator characteristic). In addition, we show that accurate orientation information greatly improved detection of conserved repeat sequence families and structure motifs. We integrated CRISPRstrand predictions into our CRISPRmap web server of CRISPR conservation and updated the latter to version 2.0.
Availability: CRISPRmap and CRISPRstrand are available at http://rna.informatik.uni-freiburg.de/CRISPRmap.
Contact: backofen@informatik.uni-freiburg.de
Supplementary information: Supplementary data are available at Bioinformatics online.
doi:10.1093/bioinformatics/btu459
PMCID: PMC4147912  PMID: 25161238
20.  CRISPR transcript processing: a mechanism for generating a large number of small interfering RNAs 
Biology Direct  2012;7:24.
Background
CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated sequences) is a recently discovered prokaryotic defense system against foreign DNA, including viruses and plasmids. CRISPR cassette is transcribed as a continuous transcript (pre-crRNA), which is processed by Cas proteins into small RNA molecules (crRNAs) that are responsible for defense against invading viruses. Experiments in E. coli report that overexpression of cas genes generates a large number of crRNAs, from only few pre-crRNAs.
Results
We here develop a minimal model of CRISPR processing, which we parameterize based on available experimental data. From the model, we show that the system can generate a large amount of crRNAs, based on only a small decrease in the amount of pre-crRNAs. The relationship between the decrease of pre-crRNAs and the increase of crRNAs corresponds to strong linear amplification. Interestingly, this strong amplification crucially depends on fast non-specific degradation of pre-crRNA by an unidentified nuclease. We show that overexpression of cas genes above a certain level does not result in further increase of crRNA, but that this saturation can be relieved if the rate of CRISPR transcription is increased. We furthermore show that a small increase of CRISPR transcription rate can substantially decrease the extent of cas gene activation necessary to achieve a desired amount of crRNA.
Conclusions
The simple mathematical model developed here is able to explain existing experimental observations on CRISPR transcript processing in Escherichia coli. The model shows that a competition between specific pre-crRNA processing and non-specific degradation determines the steady-state levels of crRNA and is responsible for strong linear amplification of crRNAs when cas genes are overexpressed. The model further shows how disappearance of only a few pre-crRNA molecules normally present in the cell can lead to a large (two orders of magnitude) increase of crRNAs upon cas overexpression. A crucial ingredient of this large increase is fast non-specific degradation by an unspecified nuclease, which suggests that a yet unidentified nuclease(s) is a major control element of CRISPR response. Transcriptional regulation may be another important control mechanism, as it can either increase the amount of generated pre-crRNA, or alter the level of cas gene activity.
Reviewers
This article was reviewed by Mikhail Gelfand, Eugene Koonin and L Aravind.
doi:10.1186/1745-6150-7-24
PMCID: PMC3537551  PMID: 22849651
CRISPR/Cas; Transcript processing; Small RNA; CRISPR expression regulation; CRISPR/Cas response
21.  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
22.  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
23.  Target Motifs Affecting Natural Immunity by a Constitutive CRISPR-Cas System in Escherichia coli 
PLoS ONE  2012;7(11):e50797.
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated (cas) genes conform the CRISPR-Cas systems of various bacteria and archaea and produce degradation of invading nucleic acids containing sequences (protospacers) that are complementary to repeat intervening spacers. It has been demonstrated that the base sequence identity of a protospacer with the cognate spacer and the presence of a protospacer adjacent motif (PAM) influence CRISPR-mediated interference efficiency. By using an original transformation assay with plasmids targeted by a resident spacer here we show that natural CRISPR-mediated immunity against invading DNA occurs in wild type Escherichia coli. Unexpectedly, the strongest activity is observed with protospacer adjoining nucleotides (interference motifs) that differ from the PAM both in sequence and location. Hence, our results document for the first time native CRISPR activity in E. coli and demonstrate that positions next to the PAM in invading DNA influence their recognition and degradation by these prokaryotic immune systems.
doi:10.1371/journal.pone.0050797
PMCID: PMC3506596  PMID: 23189210
24.  Comparative analysis of CRISPR cassettes from the human gut metagenomic contigs 
BMC Genomics  2014;15:202.
Background
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a prokaryotic adaptive defence system that provides resistance against alien replicons such as viruses and plasmids. Spacers in a CRISPR cassette confer immunity against viruses and plasmids containing regions complementary to the spacers and hence they retain a footprint of interactions between prokaryotes and their viruses in individual strains and ecosystems. The human gut is a rich habitat populated by numerous microorganisms, but a large fraction of these are unculturable and little is known about them in general and their CRISPR systems in particular.
Results
We used human gut metagenomic data from three open projects in order to characterize the composition and dynamics of CRISPR cassettes in the human-associated microbiota. Applying available CRISPR-identification algorithms and a previously designed filtering procedure to the assembled human gut metagenomic contigs, we found 388 CRISPR cassettes, 373 of which had repeats not observed previously in complete genomes or other datasets. Only 171 of 3,545 identified spacers were coupled with protospacers from the human gut metagenomic contigs. The number of matches to GenBank sequences was negligible, providing protospacers for 26 spacers.
Reconstruction of CRISPR cassettes allowed us to track the dynamics of spacer content. In agreement with other published observations we show that spacers shared by different cassettes (and hence likely older ones) tend to the trailer ends, whereas spacers with matches in the metagenomes are distributed unevenly across cassettes, demonstrating a preference to form clusters closer to the active end of a CRISPR cassette, adjacent to the leader, and hence suggesting dynamical interactions between prokaryotes and viruses in the human gut. Remarkably, spacers match protospacers in the metagenome of the same individual with frequency comparable to a random control, but may match protospacers from metagenomes of other individuals.
Conclusions
The analysis of assembled contigs is complementary to the approach based on the analysis of original reads and hence provides additional data about composition and evolution of CRISPR cassettes, revealing the dynamics of CRISPR-phage interactions in metagenomes.
doi:10.1186/1471-2164-15-202
PMCID: PMC4004331  PMID: 24628983
CRISPR; Human gut; Microbiome
25.  CRISPR/Cas systems in archaea 
Mobile Genetic Elements  2012;2(1):63-64.
CRISPR (Clustered, Regularly, Interspaced, Short, Palindromic Repeats) loci have been shown to provide prokaryotes with an adaptive immunity against viruses and plasmids. CRISPR arrays are transcribed and processed into small CRISPR RNA molecules, which base-pair with invading DNA or RNA and lead to its degradation by CRISPR-associated (Cas) protein complexes. New spacers can be acquired by active CRISPR/Cas systems, and thus the sequences of these spacers provide a record of the past “infection history” of the organism. Recently we used spacer sequences from archaeal genomes to infer gene exchange events among archaeal species and genera and to demonstrate that at least in this domain of life CRISPR indeed has an anti-viral role.
doi:10.4161/mge.19907
PMCID: PMC3383453  PMID: 22754756
CRISPR; Lateral Gene Transfer; archaea; horizontal gene transfer; viruses

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