Landes Highlights
Landes Highlights
Editor's Corner
Special focus CRISPR-Cas
Review
Diversity of CRISPR systems in the euryarchaeal Pyrococcales
Pyrococcales are members of the order Thermococcales, a group of hyperthermophilic euryarchaea that are frequently found in deep sea hydrothermal vents. Infectious genetic elements, such as plasmids and viruses, remain a threat even in this remote environment and these microorganisms have developed several ways to fight their genetic invaders. Among these are the recently discovered CRISPR systems. In this review, we have combined and condensed available information on genetic elements infecting the Thermococcales and on the multiple CRISPR systems found in the Pyrococcales to fight them. Their organization and mode of action will be presented with emphasis on the Type III-B system that is the only CRISPR system known to target RNA molecules in a process reminiscent of RNA interference. The intriguing case of Pyrococcus abyssi, which is among the rare strains to present a CRISPR system devoid of the universal cas1 and cas2 genes, is also discussed.
Review
CRISPR-mediated defense mechanisms in the hyperthermophilic archaeal genus Sulfolobus
CRISPR (clustered regularly interspaced short palindromic repeats)-mediated virus defense based on small RNAs is a hallmark of archaea and also found in many bacteria. Archaeal genomes, and in particular, organisms of the extremely thermoacidophilic genus Sulfolobus, carry extensive CRISPR loci each with dozens of sequence signatures (spacers) able to mediate targeting and degradation of complementary invading nucleic acids. The diversity of CRISPR systems and their associated protein complexes indicates an extensive functional breadth and versatility of this adaptive immune system. Sulfolobus solfataricus and S. islandicus represent two of the best characterized genetic model organisms in the archaea not only with respect to the CRISPR system. Here we address and discuss in a broader context particularly recent progress made in understanding spacer recruitment from foreign DNA, production of small RNAs, in vitro activity of CRISPR-associated protein complexes and attack of viruses and plasmids in in vivo test systems.
Review
CRISPR-Cas: Evolution of an RNA-based adaptive immunity system in prokaryotes
The CRISPR-Cas (clustered regularly interspaced short palindromic repeats, CRISPR-associated genes) is an adaptive immunity system in bacteria and archaea that functions via a distinct self-non-self recognition mechanism that is partially analogous to the mechanism of eukaryotic RNA interference (RNAi). The CRISPR-Cas system incorporates fragments of virus or plasmid DNA into the CRISPR repeat cassettes and employs the processed transcripts of these spacers as guide RNAs to cleave the cognate foreign DNA or RNA. The Cas proteins, however, are not homologous to the proteins involved in RNAi and comprise numerous, highly diverged families. The majority of the Cas proteins contain diverse variants of the RNA recognition motif (RRM), a widespread RNA-binding domain. Despite the fast evolution that is typical of the cas genes, the presence of diverse versions of the RRM in most Cas proteins provides for a simple scenario for the evolution of the three distinct types of CRISPR-cas systems. In addition to several proteins that are directly implicated in the immune response, the cas genes encode a variety of proteins that are homologous to prokaryotic toxins that typically possess nuclease activity. The predicted toxins associated with CRISPR-Cas systems include the essential Cas2 protein, proteins of COG1517 that, in addition to a ligand-binding domain and a helix-turn-helix domain, typically contain different nuclease domains and several other predicted nucleases. The tight association of the CRISPR-Cas immunity systems with predicted toxins that, upon activation, would induce dormancy or cell death suggests that adaptive immunity and dormancy/suicide response are functionally coupled. Such coupling could manifest in the persistence state being induced and potentially providing conditions for more effective action of the immune system or in cell death being triggered when immunity fails.
Brief Communication
Evidence for the widespread distribution of CRISPR-Cas system in the Phylum Cyanobacteria
Members of the phylum Cyanobacteria inhabit ecologically diverse environments. However, the CRISPR-Cas (clustered regularly interspaced short palindromic repeats, CRISPR associated genes), an extremely adaptable defense system, has not been surveyed in this phylum. We analyzed 126 cyanobacterial genomes and, surprisingly, found CRISPR-Cas in the majority except the marine subclade (Synechococcus and Prochlorococcus), in which cyanophages are a known force shaping their evolution. Multiple observations of CRISPR loci in the absence of cas1/cas2 genes may represent an early stage of losing a CRISPR-Cas locus. Our findings reveal the widespread distribution of their role in the phylum Cyanobacteria and provide a first step to systematically understanding CRISPR-Cas systems in cyanobacteria.
Technical Paper
CRISPR decoys: Competitive inhibitors of CRISPR immunity
CRISPR loci consist of an array of short repeats separated by spacer sequences that match the genome of viruses and plasmids that infect prokaryotes. Transcription of the CRISPR array generates small antisense RNAs that mediate immunity against these invaders. In recent years, there has been a notable increase in the investigation of CRISPR immunity, but studies have been restricted to organisms in which genetic manipulations are possible. Therefore, there is a need for the development of simple genetic tools that facilitate the study of this important pathway. Here we describe the use of CRISPR decoys, plasmids containing a non-transcribed repeat-spacer unit that disrupt CRISPR immunity. We show that decoys abrogate immunity against conjugation in S. epidermidis to levels comparable to a CRISPR deletion mutant. This technique can be used to generate full or spacer-specific CRISPR knockdowns in organisms in which decoy plasmids can be introduced.
Research Paper
Comparative analysis of Cas6b processing and CRISPR RNA stability
The prokaryotic antiviral defense systems CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) employs short crRNAs (CRISPR RNAs) to target invading viral nucleic acids. A short spacer sequence of these crRNAs can be derived from a viral genome and recognizes a reoccurring attack of a virus via base complementarity. We analyzed the effect of spacer sequences on the maturation of crRNAs of the subtype I-B Methanococcus maripaludis C5 CRISPR cluster. The responsible endonuclease, termed Cas6b, bound non-hydrolyzable repeat RNA as a dimer and mature crRNA as a monomer. Comparative analysis of Cas6b processing of individual spacer-repeat-spacer RNA substrates and crRNA stability revealed the potential influence of spacer sequence and length on these parameters. Correlation of these observations with the variable abundance of crRNAs visualized by deep-sequencing analyses is discussed. Finally, insertion of spacer and repeat sequences with archaeal poly-T termination signals is suggested to be prevented in archaeal CRISPR/Cas systems.
Research Paper
RcsB-BglJ-mediated activation of Cascade operon does not induce the maturation of CRISPR RNAs in E. coli K12
Prokaryotic immunity against foreign nucleic acids mediated by clustered, regularly interspaced, short palindromic repeats (CRISPR) depends on the expression of the CRISPR-associated (Cas) proteins and the formation of small CRISPR RNAs (crRNAs). The crRNA-loaded Cas ribonucleoprotein complexes convey the specific recognition and inactivation of target nucleic acids. In E. coli K12, the maturation of crRNAs and the interference with target DNA is performed by the Cascade complex. The transcription of the Cascade operon is tightly repressed through H-NS-dependent inhibition of the Pcas promoter. Elevated levels of the LysR-type regulator LeuO induce the Pcas promoter and concomitantly activate the CRISPR-mediated immunity against phages. Here, we show that the Pcas promoter can also be induced by constitutive expression of the regulator BglJ. This activation is LeuO-dependent as heterodimers of BglJ and RcsB activate leuO transcription. Each transcription factor, LeuO or BglJ, induced the transcription of the Cascade genes to comparable amounts. However, the maturation of the crRNAs was activated in LeuO but not in BglJ-expressing cells. Studies on CRISPR promoter activities, transcript stabilities, crRNA processing and Cascade protein levels were performed to answer the question why crRNA maturation is defective in BglJ-expressing cells. Our results demonstrate that the activation of Cascade gene transcription is necessary but not sufficient to turn on the CRISPR-mediated immunity and suggest a more complex regulation of the type I-E CRISPR-Cas system in E. coli.
Research Paper
High-throughput analysis of type I-E CRISPR/Cas spacer acquisition in E. coli
In Escherichia coli, the acquisition of new CRISPR spacers is strongly stimulated by a priming interaction between a spacer in CRISPR RNA and a protospacer in foreign DNA. Priming also leads to a pronounced bias in DNA strand from which new spacers are selected. Here, ca. 200,000 spacers acquired during E. coli type I-E CRISPR/Cas-driven plasmid elimination were analyzed. Analysis of positions of plasmid protospacers from which newly acquired spacers have been derived is inconsistent with spacer acquisition machinery sliding along the target DNA as the primary mechanism responsible for strand bias during primed spacer acquisition. Most protospacers that served as donors of newly acquired spacers during primed spacer acquisition had an AAG protospacer adjacent motif, PAM. Yet, the introduction of multiple AAG sequences in the target DNA had no effect on the choice of protospacers used for adaptation, which again is inconsistent with the sliding mechanism. Despite a strong preference for an AAG PAM during CRISPR adaptation, the AAG (and CTT) triplets do not appear to be avoided in known E. coli phages. Likewise, PAM sequences are not avoided in Streptococcus thermophilus phages, indicating that CRISPR/Cas systems may not have been a strong factor in shaping host-virus interactions.
Research Paper
The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems
CRISPR-Cas is a rapidly evolving RNA-mediated adaptive immune system that protects bacteria and archaea against mobile genetic elements. The system relies on the activity of short mature CRISPR RNAs (crRNAs) that guide Cas protein(s) to silence invading nucleic acids. A set of CRISPR-Cas, type II, requires a trans-activating small RNA, tracrRNA, for maturation of precursor crRNA (pre-crRNA) and interference with invading sequences. Following co-processing of tracrRNA and pre-crRNA by RNase III, dual-tracrRNA:crRNA guides the CRISPR-associated endonuclease Cas9 (Csn1) to cleave site-specifically cognate target DNA. Here, we screened available genomes for type II CRISPR-Cas loci by searching for Cas9 orthologs. We analyzed 75 representative loci, and for 56 of them we predicted novel tracrRNA orthologs. Our analysis demonstrates a high diversity in cas operon architecture and position of the tracrRNA gene within CRISPR-Cas loci. We observed a correlation between locus heterogeneity and Cas9 sequence diversity, resulting in the identification of various type II CRISPR-Cas subgroups. We validated the expression and co-processing of predicted tracrRNAs and pre-crRNAs by RNA sequencing in five bacterial species. This study reveals tracrRNA family as an atypical, small RNA family with no obvious conservation of structure, sequence or localization within type II CRISPR-Cas loci. The tracrRNA family is however characterized by the conserved feature to base-pair to cognate pre-crRNA repeats, an essential function for crRNA maturation and DNA silencing by dual-RNA:Cas9. The large panel of tracrRNA and Cas9 ortholog sequences should constitute a useful database to improve the design of RNA-programmable Cas9 as genome editing tool.
Research Paper
Genetic determinants of PAM-dependent DNA targeting and pre-crRNA processing in Sulfolobus islandicus
Bacteria and Archaea encode clustered, regularly interspaced, short palindromic repeat (CRISPR) systems to confer adaptive immunity to invasive viruses and plasmids. Recent studies of CRISPR systems revealed that diverse CRISPR-associated (Cas) interference modules often coexist in different organisms but functions of cas genes have not been dissected in any of these systems. The crenarchaeon Sulfolobus islandicus encodes three distinct CRISPR interference modules, including a type IA system and two type IIIB systems: Cmr-α and Cmr-β. To study the genetic determinants of protospacer-adjacent motif (PAM)-dependent DNA targeting activity and mature CRISPR RNA (crRNA) production in this organism, mutants deleting individual genes of the type IA system or removing each of other Cas modules were constructed. Characterization of these mutants revealed that Cas7, Cas5, Cas6, Cas3′ and Cas3” are essential for PAM-dependent DNA targeting activity, whereas Csa5, along with all other Cas modules, is dispensable for the targeting in the crenarchaeon. Cas6 is implicated as the only enzyme for pre-crRNA processing and the crRNA maturation is independent of the DNA targeting activity. Importantly, we show that Cas7 and Cas5 are essential for stabilizing the processing intermediates and mature crRNAs, respectively, and that depleting the helicase or nuclease domain of Cas3 leads to the accumulation of processing intermediates. This demonstrates that in addition to Cas6, other Cas proteins of an archaeal type IA system also contribute to crRNA processing.
Research Paper
CRISPR-Cas systems preferentially target the leading regions of MOBF conjugative plasmids
Most prokaryotes contain CRISPR-Cas immune systems that provide protection against mobile genetic elements. We have focused on the ability of CRISPR-Cas to block plasmid conjugation, and analyzed the position of target sequences (protospacers) on conjugative plasmids. The analysis reveals that protospacers are non-uniformly distributed over plasmid regions in a pattern that is determined by the plasmid’s mobilization type (MOB). While MOBP plasmids are most frequently targeted in the region entering the recipient cell last (lagging region), MOBF plasmids are mostly targeted in the region entering the recipient cell first (leading region). To explain this protospacer distribution bias, we propose two mutually non-exclusive hypotheses: (1) spacers are acquired more frequently from either the leading or lagging region depending on the MOB type (2) CRISPR-interference is more efficient when spacers target these preferred regions. To test the latter hypothesis, we analyzed Type I-E CRISPR-interference against MOBF prototype plasmid F in Escherichia coli. Our results show that plasmid conjugation is effectively inhibited, but the level of immunity is not affected by targeting the plasmid in the leading or lagging region. Moreover, CRISPR-immunity levels do not depend on whether the incoming single-stranded plasmid DNA, or the DNA strand synthesized in the recipient is targeted. Our findings indicate that single-stranded DNA may not be a target for Type I-E CRISPR-Cas systems, and suggest that the protospacer distribution bias might be due to spacer acquisition preferences.
Research Paper
Structure of the archaeal Cascade subunit Csa5: Relating the small subunits of CRISPR effector complexes
The Cascade complex for CRISPR-mediated antiviral immunity uses CRISPR RNA (crRNA) to target invading DNA species from mobile elements such as viruses, leading to their destruction. The core of the Cascade effector complex consists of the Cas5 and Cas7 subunits, which are widely conserved in prokaryotes. Cas7 binds crRNA and forms the helical backbone of Cascade. Many archaea encode a version of the Cascade complex (denoted Type I-A) that includes a Csa5 (or small) subunit, which interacts weakly with the core proteins. Here, we report the crystal structure of the Csa5 protein from Sulfolobus solfataricus. Csa5 comprises a conserved α-helical domain with a small insertion consisting of a weakly conserved β-strand domain. In the crystal, the Csa5 monomers have multimerized into infinite helical threads. At each interface is a strictly conserved intersubunit salt bridge, deletion of which disrupts multimerization. Structural analysis indicates a shared evolutionary history among the small subunits of the CRISPR effector complexes. The same α-helical domain is found in the C-terminal domain of Cse2 (from Type I-E Cascade), while the N-terminal domain of Cse2 is found in Cmr5 of the CMR (Type III-B) effector complex. As Cmr5 shares no match with Csa5, two possibilities present themselves: selective domain loss from an ancestral Cse2 to create two new subfamilies or domain fusion of two separate families to create a new Cse2 family. A definitive answer awaits structural studies of further small subunits from other CRISPR effector complexes.
Research Paper
Cas3 stimulates runaway replication of a ColE1 plasmid in Escherichia coli and antagonises RNaseHI
Cas3 nuclease-helicase is part of CRISPR immunity systems in many bacteria and archaea. In type I CRISPR, Cas3 nuclease degrades invader DNA that has been base-paired to crRNA as an R-loop within a “Cascade” complex. An R-loop is a DNA-RNA hybrid that includes a displaced single-strand DNA loop. Purified Cas3 from E. coli and the archaeon M. thermautrophicus can process R-loops without DNA/RNA sequence specificity and without Cascade. This has potential to affect other aspects of microbial biology that involve R-loops. Regulatory RNAs and host cell proteins modulate replication of ColE1 plasmids (e.g., pUC) from R-loop primers. We observed that Cas3 could override endogenous control of a ColE1 replicon, stimulating uncontrolled (“runaway”) replication and resulting in much higher plasmid yields. This effect was absent when using helicase-defective Cas3 (Cas3K320L) or a non-ColE1 plasmid, and was dependent on RNaseHI. Cas3 also promoted formation of plasmid multimers or concatemers, a phenotype consistent with deregulated ColE1 replication and typical of cells lacking RNaseHI. These effects of Cas3 on ColE1 plasmids are inconsistent with it unwinding R-loops in vivo, at least in this assay. We discuss a model of how Cas3 might be able to regulate RNA molecules in vivo, unless it is targeted to CRISPR defense by Cascade, or kept in check by RecG and RNaseHI.
Research Paper
Two CRISPR-Cas systems in Methanosarcina mazei strain Gö1 display common processing features despite belonging to different types I and III
The clustered regularly interspaced short palindromic repeats (CRISPR) system represents a highly adaptive and heritable defense system against foreign nucleic acids in bacteria and archaea. We analyzed the two CRISPR-Cas systems in Methanosarcina mazei strain Gö1. Although belonging to different subtypes (I-B and III-B), the leaders and repeats of both loci are nearly identical. Also, despite many point mutations in each array, a common hairpin motif was identified in the repeats by a bioinformatics analysis and in vitro structural probing. The expression and maturation of CRISPR-derived RNAs (crRNAs) were studied in vitro and in vivo. Both respective potential Cas6b-type endonucleases were purified and their activity tested in vitro. Each protein showed significant activity and could cleave both repeats at the same processing site. Cas6b of subtype III-B, however, was significantly more efficient in its cleavage activity compared with Cas6b of subtype I-B. Northern blot and differential RNAseq analyses were performed to investigate in vivo transcription and maturation of crRNAs, revealing generally very low expression of both systems, whereas significant induction at high NaCl concentrations was observed. crRNAs derived proximal to the leader were generally more abundant than distal ones and in vivo processing sites were clarified for both loci, confirming the previously well-established 8 nt 5′ repeat tags. The 3′-ends were more diverse, but generally ended in a prefix of the following repeat sequence (3′-tag). The analysis further revealed a 5′-hydroxy and 3′-phosphate termini architecture of small crRNAs specific for cleavage products of Cas6 endonucleases from type I-E and I-F and type III-B.
Research Paper
CRISPR-spacer integration reporter plasmids reveal distinct genuine acquisition specificities among CRISPR-Cas I-E variants of Escherichia coli
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.
Research Paper
Snapshot of haloarchaeal tailed virus genomes
The complete genome sequences of archaeal tailed viruses are currently highly underrepresented in sequence databases. Here, we report the genomic sequences of 10 new tailed viruses infecting different haloarchaeal hosts. Among these, only two viral genomes are closely related to each other and to previously described haloviruses HF1 and HF2. The approximately 760 kb of new genomic sequences in total shows no matches to CRISPR/Cas spacer sequences in haloarchaeal host genomes. Despite their high divergence, we were able to identify virion structural and assembly genes as well as genes coding for DNA and RNA metabolic functions. Interestingly, we identified many genes and genomic features that are shared with tailed bacteriophages, consistent with the hypothesis that haloarchaeal and bacterial tailed viruses share common ancestry, and that a viral lineage containing archaeal viruses, bacteriophages and eukaryotic viruses predates the division of the three major domains of non-viral life. However, as in tailed viruses in general and in haloarchaeal tailed viruses in particular, there are still a considerable number of predicted genes of unknown function.
Research Paper
CRISPRTarget: Bioinformatic prediction and analysis of crRNA targets
The bacterial and archaeal CRISPR/Cas adaptive immune system targets specific protospacer nucleotide sequences in invading organisms. This requires base pairing between processed CRISPR RNA and the target protospacer. For type I and II CRISPR/Cas systems, protospacer adjacent motifs (PAM) are essential for target recognition, and for type III, mismatches in the flanking sequences are important in the antiviral response. In this study, we examine the properties of each class of CRISPR. We use this information to provide a tool (CRISPRTarget) that predicts the most likely targets of CRISPR RNAs (http://bioanalysis.otago.ac.nz/CRISPRTarget). This can be used to discover targets in newly sequenced genomic or metagenomic data. To test its utility, we discover features and targets of well-characterized Streptococcus thermophilus and Sulfolobus solfataricus type II and III CRISPR/Cas systems. Finally, in Pectobacterium species, we identify new CRISPR targets and propose a model of temperate phage exposure and subsequent inhibition by the type I CRISPR/Cas systems.
Research Paper
Programmable plasmid interference by the CRISPR-Cas system in Thermococcus kodakarensis
CRISPR-Cas systems are RNA-guided immune systems that protect prokaryotes against viruses and other invaders. The CRISPR locus encodes crRNAs that recognize invading nucleic acid sequences and trigger silencing by the associated Cas proteins. There are multiple CRISPR-Cas systems with distinct compositions and mechanistic processes. Thermococcus kodakarensis (Tko) is a hyperthermophilic euryarchaeon that has both a Type I-A Csa and a Type I-B Cst CRISPR-Cas system. We have analyzed the expression and composition of crRNAs from the three CRISPRs in Tko by RNA deep sequencing and northern analysis. Our results indicate that crRNAs associated with these two CRISPR-Cas systems include an 8-nucleotide conserved sequence tag at the 5′ end. We challenged Tko with plasmid invaders containing sequences targeted by endogenous crRNAs and observed active CRISPR-Cas-mediated silencing. Plasmid silencing was dependent on complementarity with a crRNA as well as on a sequence element found immediately adjacent to the crRNA recognition site in the target termed the PAM (protospacer adjacent motif). Silencing occurred independently of the orientation of the target sequence in the plasmid, and appears to occur at the DNA level, presumably via DNA degradation. In addition, we have directed silencing of an invader plasmid by genetically engineering the chromosomal CRISPR locus to express customized crRNAs directed against the plasmid. Our results support CRISPR engineering as a feasible approach to develop prokaryotic strains that are resistant to infection for use in industry.
Research Paper
crRNA and tracrRNA guide Cas9-mediated DNA interference in Streptococcus thermophilus
The Cas9-crRNA complex of the Streptococcus thermophilus DGCC7710 CRISPR3-Cas system functions as an RNA-guided endonuclease with crRNA-directed target sequence recognition and protein-mediated DNA cleavage. We show here that an additional RNA molecule, tracrRNA (trans-activating CRISPR RNA), co-purifies with the Cas9 protein isolated from the heterologous E. coli strain carrying the S. thermophilus DGCC7710 CRISPR3-Cas system. We provide experimental evidence that tracrRNA is required for Cas9-mediated DNA interference both in vitro and in vivo. We show that Cas9 specifically promotes duplex formation between the precursor crRNA (pre-crRNA) transcript and tracrRNA, in vitro. Furthermore, the housekeeping RNase III contributes to primary pre-crRNA-tracrRNA duplex cleavage for mature crRNA biogenesis. RNase III, however, is not required in the processing of a short pre-crRNA transcribed from a minimal CRISPR array containing a single spacer. Finally, we show that an in vitro-assembled ternary Cas9-crRNA-tracrRNA complex cleaves DNA. This study further specifies the molecular basis for crRNA-based re-programming of Cas9 to specifically cleave any target DNA sequence for precise genome surgery. The processes for crRNA maturation and effector complex assembly established here will contribute to the further development of the Cas9 re-programmable system for genome editing applications.
Research Paper
Adaptation and modification of three CRISPR loci in two closely related cyanobacteria
An RNA-based screen was performed to reveal a possible evolutionary scenario for the CRISPR-Cas systems in two cyanobacterial model strains. Following the analysis of a draft genome sequence of Synechocystis sp PCC6714, three different CRISPR-Cas systems were characterized that have different degrees of relatedness to another three CRISPR-Cas systems in Synechocystis sp PCC6803. A subtype III-B system was identified that is extremely conserved between both strains. Strong signals in northern hybridizations and the presence of different spacers (but identical repeats) indicated this system to be active, despite the absence of a known endonuclease candidate gene involved in the maturation of its crRNAs in the two strains. The other two systems were found to differ significantly from each other, with different sets of repeat-spacer arrays and different Cas genes. In view of the otherwise very close relatedness of the two analyzed strains, this is suggestive of an unknown mechanism involved in the replacement of CRISPR-Cas cassettes as a whole. Further RNA analyses revealed the accumulation of crRNAs to be impacted by environmental conditions critical for photoautotropic growth. All six systems are associated with a gene for a possible transcriptional repressor. Indeed, we identified one of these genes, sll7009, as encoding a negative regulator specific for the CRISPR1 subtype I-D system in Synechocystis sp PCC6803.
Research Paper
Essential requirements for the detection and degradation of invaders by the Haloferax volcanii CRISPR/Cas system I-B
To fend off foreign genetic elements, prokaryotes have developed several defense systems. The most recently discovered defense system, CRISPR/Cas, is sequence-specific, adaptive and heritable. The two central components of this system are the Cas proteins and the CRISPR RNA. The latter consists of repeat sequences that are interspersed with spacer sequences. The CRISPR locus is transcribed into a precursor RNA that is subsequently processed into short crRNAs. CRISPR/Cas systems have been identified in bacteria and archaea, and data show that many variations of this system exist. We analyzed the requirements for a successful defense reaction in the halophilic archaeon Haloferax volcanii. Haloferax encodes a CRISPR/Cas system of the I-B subtype, about which very little is known. Analysis of the mature crRNAs revealed that they contain a spacer as their central element, which is preceded by an eight-nucleotide-long 5′ handle that originates from the upstream repeat. The repeat sequences have the potential to fold into a minimal stem loop. Sequencing of the crRNA population indicated that not all of the spacers that are encoded by the three CRISPR loci are present in the same abundance. By challenging Haloferax with an invader plasmid, we demonstrated that the interaction of the crRNA with the invader DNA requires a 10-nucleotide-long seed sequence. In addition, we found that not all of the crRNAs from the three CRISPR loci are effective at triggering the degradation of invader plasmids. The interference does not seem to be influenced by the copy number of the invader plasmid.
Research Paper
Novel insights into gene regulation of the rudivirus SIRV2 infecting Sulfolobus cells
Microarray analysis of infection by a lytic Sulfolobus rudivirus, SIRV2, revealed both the temporal expression of viral genes and the differential regulation of host genes. A highly susceptible strain derived from Sulfolobus solfataricus P2 with a large genomic deletion spanning CRISPR clusters A to D was infected with SIRV2, and subjected to a microarray analysis. Transcripts from a few viral genes were detected at 15 min post-infection and all except one were expressed within 2 h. The earliest expressed genes were located mainly at the termini of the linear viral genome while later expressed genes were concentrated in the central region. Timing of the expression correlated with the known or predicted functions of the viral gene products and, thus, should facilitate functional characterization of many hypothetical viral genes. Evaluation of the microarray data with quantitative reverse-transcription PCR analyses of a few selected viral genes revealed a good correlation between the two methods. Expression of about 3,000 host genes was examined. Seventy-two were downregulated > 2-fold that were mainly associated with stress response and vesicle formation, as well as chromosome structure maintenance, which appears to contribute to host chromosome degradation and cellular collapse. A further 76 host genes were upregulated > 2-fold and they were dominated by genes associated with metabolism and membrane transport, including phosphate transport and DNA precursor synthesis. The altered transcriptional patterns suggest that the virus reprograms the host cellular machinery to facilitate its own DNA replication and to inhibit cellular processes required for defense against viruses.
Commentary
In defense of phage: Viral suppressors of CRISPR-mediated adaptive immunity in bacteria
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.
Commentary
Protospacer recognition motifs: Mixed identities and functional diversity
Protospacer adjacent motifs (PAMs) were originally characterized for CRISPR-Cas systems that were classified on the basis of their CRISPR repeat sequences. A few short 2–5 bp sequences were identified adjacent to one end of the protospacers. Experimental and bioinformatical results linked the motif to the excision of protospacers and their insertion into CRISPR loci. Subsequently, evidence accumulated from different virus- and plasmid-targeting assays, suggesting that these motifs were also recognized during DNA interference, at least for the recently classified type I and type II CRISPR-based systems. The two processes, spacer acquisition and protospacer interference, employ different molecular mechanisms, and there is increasing evidence to suggest that the sequence motifs that are recognized, while overlapping, are unlikely to be identical. In this article, we consider the properties of PAM sequences and summarize the evidence for their dual functional roles. It is proposed to use the terms protospacer associated motif (PAM) for the conserved DNA sequence and to employ spacer acqusition motif (SAM) and target interference motif (TIM), respectively, for acquisition and interference recognition sites.
Commentary
Holding a grudge: Persisting anti-phage CRISPR immunity in multiple human gut microbiomes
The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) system of bacteria and archaea constitutes a mechanism of acquired adaptive immunity against phages, which is based on genome-encoded markers of previously infecting phage sequences (“spacers”). As a repository of phage sequences, these spacers make the system particularly suitable for elucidating phage-bacteria interactions in metagenomic studies. Recent metagenomic analyses of CRISPRs associated with the human microbiome intriguingly revealed conserved “memory spacers” shared by bacteria in multiple unrelated, geographically separated individuals. Here, we discuss possible avenues for explaining this phenomenon by integrating insights from CRISPR biology and phage-bacteria ecology, with a special focus on the human gut. We further explore the growing body of evidence for the role of CRISPR/Cas in regulating the interplay between bacteria and lysogenic phages, which may be intimately related to the presence of memory spacers and sheds new light on the multifaceted biological and ecological modes of action of CRISPR/Cas.
