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.
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.
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.
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Using the hyperthermophile Pyrococcus furiosus, we have delineated several key steps in CRISPR (clustered regularly interspaced short palindromic repeats)–Cas (CRISPR-associated) invader defence pathways. P. furiosus has seven transcriptionally active CRISPR loci that together encode a total of 200 crRNAs (CRISPR RNAs). The 27 Cas proteins in this organism represent three distinct pathways and are primarily encoded in two large gene clusters. The Cas6 protein dices CRISPR locus transcripts to generate individual invader-targeting crRNAs. The mature crRNAs include a signature sequence element (the 5′ tag) derived from the CRISPR locus repeat sequence that is important for function. crRNAs are tailored into distinct species and integrated into three distinct crRNA–Cas protein complexes that are all candidate effector complexes. The complex formed by the Cmr [Cas module RAMP (repeat-associated mysterious proteins)] (subtype III-B) proteins cleaves complementary target RNAs and can be programmed to cleave novel target RNAs in a prokaryotic RNAi-like manner. Evidence suggests that the other two CRISPR–Cas systems in P. furiosus, Csa (Cas subtype Apern) (subtype I-A) and Cst (Cas subtype Tneap) (subtype I-B), target invaders at the DNA level. Studies of the CRISPR–Cas systems from P. furiosus are yielding fundamental knowledge of mechanisms of crRNA biogenesis and silencing for three of the diverse CRISPR–Cas pathways, and reveal that organisms such as P. furiosus possess an arsenal of multiple RNA-guided mechanisms to resist diverse invaders. Our knowledge of the fascinating CRISPR–Cas pathways is leading in turn to our ability to co-opt these systems for exciting new biomedical and biotechnological applications.
clustered regularly interspaced short palindromic repeats (CRISPR); CRISPR-associated (Cas); non-coding RNA; prokaryotic immunity; Pyrococcus furiosus; virus
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.
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.
The CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) found in prokaryotic genomes confer small RNA-mediated protection against viruses and other invaders. CRISPR loci contain iterations of a short repeat sequence alternating with small segments of varying invader-derived sequences. Distinct families of CRISPR-associated Cas proteins function to cleave within the repeat sequence of CRISPR transcripts and produce the individual invader-targeting crRNAs. Here we report the crystal structure of Pyrococcus furiosus Cas6 bound with a repeat RNA at 3.2 Å resolution. In contrast to other Cas families of endonucleases, Cas6 clasps nucleotides 2–9 of the repeat RNA using its two ferredoxin-like domains, and the enzyme-anchored 5’ end tethers the distal cleavage site of the RNA between nucleotides 22 and 23 to the predicted enzyme active site on the opposite side of the ferrodoxin-like domains. Our findings suggest a wrap-around mechanism for CRISPR RNA recognition and cleavage by Cas6 and related processing endonucleases.
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.
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.
Clustered regularly interspaced short palindromic repeat (CRISPR) loci and CRISPR-associated (Cas) proteins form an adaptive immune system that protects prokaryotes against plasmids and viruses. The Cmr complex, a type III-B effector complex, uses the CRISPR RNA (crRNA) as a guide to target RNA. Here, we show that the Cmr complex of Pyrococcus furiosus cleaves RNA at multiple sites that are 6 nt apart and are positioned relative to the 5′-end of the crRNA. We identified Cmr4 as the slicer and determined its crystal structure at 2.8 Å resolution. In the crystal, Cmr4 forms a helical filament that most likely reflects its structural organization in the Cmr complex. The putative active site is located at the inner surface of the filament where the guide and substrate RNA are thought to bind. The filament structure of Cmr4 accounts for multiple periodic cleavage sites on the substrate. Our study provides new insights into the structure and mechanism of the RNA-targeting Cmr complex.
The recently discovered clustered regularly interspaced short palindromic repeat (CRISPR)-mediated virus defense represents an adaptive immune system in many bacteria and archaea. Small CRISPR RNAs cause cleavage of complementary invading nucleic acids in conjunction with an associated protein or a protein complex. Here, we show CRISPR-mediated cleavage of mRNA from an invading virus in the hyperthermophilic archaeon Sulfolobus solfataricus. More than 40% of the targeted mRNA could be cleaved, as demonstrated by quantitative polymerase chain reaction. Cleavage of the mRNA was visualized by northern analyses and cleavage sites were mapped. In vitro, the same substrates were cleaved by the purified CRISPR-associated CMR complex from Sulfolobus solfataricus. The in vivo system was also re-programmed to knock down mRNA of a selected chromosomal gene (β-galactosidase) using an artificial miniCRISPR locus. With a single complementary spacer, ∼50% reduction of the targeted mRNA and of corresponding intracellular protein activity was achieved. Our results demonstrate in vivo cleavage of mRNA in a prokaryote mediated by small RNAs (i.e. analogous to RNA interference in eukaryotes) and the re-programming of the system to silence specific genes of interest.
Small RNAs target invaders for silencing in the CRISPR-Cas pathways that protect bacteria and archaea from viruses and plasmids. The CRISPR RNAs (crRNAs) contain sequence elements acquired from invaders that guide CRISPR-associated (Cas) proteins back to the complementary invading DNA or RNA. Here, we have analyzed essential features of the crRNAs associated with the Cas RAMP module (Cmr) effector complex, which cleaves targeted RNAs. We show that Cmr crRNAs contain an 8-nucleotide 5’ sequence tag (also found on crRNAs associated with other CRISPR-Cas pathways) that is critical for crRNA function and can be used to engineer crRNAs that direct cleavage of novel targets. We also present data that indicates that the Cmr complex cleaves an endogenous complementary RNA in Pyrococcus furiosus, providing direct in vivo evidence of RNA targeting by the CRISPR-Cas system. Our findings indicate that the CRISPR RNA-Cmr protein pathway may be exploited to cleave RNAs of interest.
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.
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.
The prokaryotic CRISPR (clustered regularly interspaced palindromic repeats)-associated protein, Cas9, has been widely adopted as a tool for editing, imaging, and regulating eukaryotic genomes. However, our understanding of how to select single-guide RNAs (sgRNAs) that mediate efficient Cas9 activity is incomplete, as we lack insight into how chromatin impacts Cas9 targeting. To address this gap, we analyzed large-scale genetic screens performed in human cell lines using either nuclease-active or nuclease-dead Cas9 (dCas9). We observed that highly active sgRNAs for Cas9 and dCas9 were found almost exclusively in regions of low nucleosome occupancy. In vitro experiments demonstrated that nucleosomes in fact directly impede Cas9 binding and cleavage, while chromatin remodeling can restore Cas9 access. Our results reveal a critical role of eukaryotic chromatin in dictating the targeting specificity of this transplanted bacterial enzyme, and provide rules for selecting Cas9 target sites distinct from and complementary to those based on sequence properties.
Many bacteria have a type of immune system known as CRISPR that can target and cut foreign DNA to protect it against viruses. Recently, the CRISPR system was adapted to allow scientists to easily manipulate the genome of humans and many other organisms. However, unlike the loosely organized DNA found in bacteria, the DNA that makes up the human genome is tightly packed and wrapped around complexes of proteins to form structures called nucleosomes. It was not clear whether the CRISPR system was able to effectively target the stretches of DNA in a nucleosome.
In 2013, researchers developed a modified version of CRISPR, known as CRISPR interference, to block gene activity and in 2014 used it to systematically repress many of the genes in the human genome. Now, Horlbeck, Witkowsky et al. – who include several of the researchers from the 2014 work – have analyzed existing data for a specific type of human cell grown in the laboratory and found that CRISPR interference activity was strongest in certain areas around the start of each gene. However, CRISPR interference was much weaker in other areas of genes that coincided well with stretches of DNA that are known to often be bound by nucleosomes. Nucleosomes also appeared to block CRISPR editing, although the effects were less pronounced.
Horlbeck, Witkowsky et al. then directly tested whether nucleosomes could prevent the CRISPR system from binding or modifying the DNA. When the individual components were mixed in test tubes, the CRISPR system could readily target “naked” DNA. However, it could not access nucleosome-bound DNA, unless an enzyme that can move nucleosomes along the DNA in the human genome was also added to the mix. These findings suggest one way that CRISPR can manipulate much of the human genome despite the widespread presence of nucleosomes. Future work will now aim to develop computational methods that take the positions of nucleosomes into account when picking DNA sites to target with CRISPR.
CRISPR/Cas; nucleosomes; chromatin; Human
CRISPR (cluster of regularly interspaced palindromic repeats) is a prokaryotic adaptive defence system, providing immunity against mobile genetic elements such as viruses. Genomically encoded crRNA (CRISPR RNA) is used by Cas (CRISPR-associated) proteins to target and subsequently degrade nucleic acids of invading entities in a sequence-dependent manner. The process is known as ‘interference’. In the present review we cover recent progress on the structural biology of the CRISPR/Cas system, focusing on the Cas proteins and complexes that catalyse crRNA biogenesis and interference. Structural studies have helped in the elucidation of key mechanisms, including the recognition and cleavage of crRNA by the Cas6 and Cas5 proteins, where remarkable diversity at the level of both substrate recognition and catalysis has become apparent. The RNA-binding RAMP (repeat-associated mysterious protein) domain is present in the Cas5, Cas6, Cas7 and Cmr3 protein families and RAMP-like domains are found in Cas2 and Cas10. Structural analysis has also revealed an evolutionary link between the small subunits of the type I and type III-B interference complexes. Future studies of the interference complexes and their constituent components will transform our understanding of the system.
antiviral defence; cluster of regularly interspaced palindromic repeats (CRISPR); crystallography; evolution; protein structure; repeat-associated mysterious protein (RAMP); BhCas5c, Bacillus halodurans Cas5c; CRISPR, cluster of regularly interspaced palindromic repeats; Cas, CRISPR-associated; Cascade, CRISPR-associated complex for antiviral defence; crRNA, CRISPR RNA; dsDNA, double-stranded DNA; EcoCas3, Escherichia coli Cas3; EM, electron microscopy; HD, histidine–aspartate; MjaCas3″, Methanocaldococcus jannaschii Cas3″; PaCas6f, Pseudomonas aeruginosa Cas6f; PAM, protospacer adjacent motif; PfuCas, Pyrococcus furiosus Cas; pre-crRNA, precursor crRNA; RAMP, repeat-associated mysterious protein; RRM, RNA recognition motif; ssDNA, single-stranded DNA; SsoCas, Sulfolobus solfataricus Cas; ssRNA, single-stranded RNA; SthCas3, Streptococcus thermophilus Cas3; tracrRNA, trans-activating crRNA; TtCas, Thermus thermophilus Cas
CRISPR–Cas systems silence plasmids and viruses in prokaryotes. CRISPR–Cas effector complexes contain CRISPR RNAs (crRNAs) that include sequences captured from invaders and direct CRISPR-associated (Cas) proteins to destroy corresponding invader nucleic acids. Pyrococcus furiosus (Pfu) harbors three CRISPR–Cas immune systems: a Cst (Type I-G) system with an associated Cmr (Type III-B) module at one locus, and a partial Csa (Type I-A) module (lacking known invader sequence acquisition and crRNA processing genes) at another locus. The Pfu Cmr complex cleaves complementary target RNAs, and Csa systems have been shown to target DNA, while the mechanism by which Cst complexes silence invaders is unknown. In this study, we investigated the function of the Cst as well as Csa system in Pfu strains harboring a single CRISPR–Cas system. Plasmid transformation assays revealed that the Cst and Csa systems both function by DNA silencing and utilize similar flanking sequence information (PAMs) to identify invader DNA. Silencing by each system specifically requires its associated Cas3 nuclease. crRNAs from the 7 shared CRISPR loci in Pfu are processed for use by all 3 effector complexes, and Northern analysis revealed that individual effector complexes dictate the profile of mature crRNA species that is generated.
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.
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.
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.
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.
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.
Background: The Cas6 protein is required for generating crRNAs in CRISPR-Cas I and III systems.
Results: The Cas6 protein is necessary for crRNA production but not sufficient for crRNA maintenance in Haloferax.
Conclusion: A Cascade-like complex is required in the type I-B system for a stable crRNA population.
Significance: The CRISPR-Cas system I-B has a similar Cascade complex like types I-A and I-E.
The clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR-Cas) system is a prokaryotic defense mechanism against foreign genetic elements. A plethora of CRISPR-Cas versions exist, with more than 40 different Cas protein families and several different molecular approaches to fight the invading DNA. One of the key players in the system is the CRISPR-derived RNA (crRNA), which directs the invader-degrading Cas protein complex to the invader. The CRISPR-Cas types I and III use the Cas6 protein to generate mature crRNAs. Here, we show that the Cas6 protein is necessary for crRNA production but that additional Cas proteins that form a CRISPR-associated complex for antiviral defense (Cascade)-like complex are needed for crRNA stability in the CRISPR-Cas type I-B system in Haloferax volcanii in vivo. Deletion of the cas6 gene results in the loss of mature crRNAs and interference. However, cells that have the complete cas gene cluster (cas1–8b) removed and are transformed with the cas6 gene are not able to produce and stably maintain mature crRNAs. crRNA production and stability is rescued only if cas5, -6, and -7 are present. Mutational analysis of the cas6 gene reveals three amino acids (His-41, Gly-256, and Gly-258) that are essential for pre-crRNA cleavage, whereas the mutation of two amino acids (Ser-115 and Ser-224) leads to an increase of crRNA amounts. This is the first systematic in vivo analysis of Cas6 protein variants. In addition, we show that the H. volcanii I-B system contains a Cascade-like complex with a Cas7, Cas5, and Cas6 core that protects the crRNA.
Archaea; Microbiology; Molecular Biology; Molecular Genetics; Protein Complexes; CRISPR/Cas; Cas6; Haloferax volcanii; crRNA; Type I-B
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.
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.
The classical and El Tor biotypes of Vibrio cholerae serogroup O1, the etiological agent of cholera, are responsible for the sixth and seventh (current) pandemics, respectively. A genomic island (GI), GI-24, previously identified in a classical biotype strain of V. cholerae, is predicted to encode clustered regularly interspaced short palindromic repeat (CRISPR)-associated proteins (Cas proteins); however, experimental evidence in support of CRISPR activity in V. cholerae has not been documented. Here, we show that CRISPR-Cas is ubiquitous in strains of the classical biotype but excluded from strains of the El Tor biotype. We also provide in silico evidence to suggest that CRISPR-Cas actively contributes to phage resistance in classical strains. We demonstrate that transfer of GI-24 to V. cholerae El Tor via natural transformation enables CRISPR-Cas-mediated resistance to bacteriophage CP-T1 under laboratory conditions. To elucidate the sequence requirements of this type I-E CRISPR-Cas system, we engineered a plasmid-based system allowing the directed targeting of a region of interest. Through screening for phage mutants that escape CRISPR-Cas-mediated resistance, we show that CRISPR targets must be accompanied by a 3′ TT protospacer-adjacent motif (PAM) for efficient interference. Finally, we demonstrate that efficient editing of V. cholerae lytic phage genomes can be performed by simultaneously introducing an editing template that allows homologous recombination and escape from CRISPR-Cas targeting.
IMPORTANCE Cholera, caused by the facultative pathogen Vibrio cholerae, remains a serious public health threat. Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) provide prokaryotes with sequence-specific protection from invading nucleic acids, including bacteriophages. In this work, we show that one genomic feature differentiating sixth pandemic (classical biotype) strains from seventh pandemic (El Tor biotype) strains is the presence of a CRISPR-Cas system in the classical biotype. We demonstrate that the CRISPR-Cas system from a classical biotype strain can be transferred to a V. cholerae El Tor biotype strain and that it is functional in providing resistance to phage infection. Finally, we show that this CRISPR-Cas system can be used as an efficient tool for the editing of V. cholerae lytic phage genomes.
Prokaryotes have evolved several defence mechanisms to protect themselves from viral predators. Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated proteins (Cas) display a prokaryotic adaptive immune system that memorizes previous infections by integrating short sequences of invading genomes—termed spacers—into the CRISPR locus. The spacers interspaced with repeats are expressed as small guide CRISPR RNAs (crRNAs) that are employed by Cas proteins to target invaders sequence-specifically upon a reoccurring infection. The ability of the minimal CRISPR-Cas9 system to target DNA sequences using programmable RNAs has opened new avenues in genome editing in a broad range of cells and organisms with high potential in therapeutical applications. While numerous scientific studies have shed light on the biochemical processes behind CRISPR-Cas systems, several aspects of the immunity steps, however, still lack sufficient understanding. This review summarizes major discoveries in the CRISPR-Cas field, discusses the role of CRISPR-Cas in prokaryotic immunity and other physiological properties, and describes applications of the system as a DNA editing technology and antimicrobial agent.
This article is part of the themed issue ‘The new bacteriology’.
CRISPR; Cas9; bacteriophage; genome editing
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”.
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.
Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) genes are present in many bacterial and archaeal genomes. Since the discovery of the typical CRISPR loci in the 1980s, well before their physiological role was revealed, their variable sequences have been used as a complementary typing tool in diagnostic, epidemiologic, and evolutionary analyses of prokaryotic strains. The discovery that CRISPR spacers are often identical to sequence fragments of mobile genetic elements was a major breakthrough that eventually led to the elucidation of CRISPR-Cas as an adaptive immunity system. Key elements of this unique prokaryotic defense system are small CRISPR RNAs that guide nucleases to complementary target nucleic acids of invading viruses and plasmids, generally followed by the degradation of the invader. In addition, several recent studies have pointed at direct links of CRISPR-Cas to regulation of a range of stress-related phenomena. An interesting example concerns a pathogenic bacterium that possesses a CRISPR-associated ribonucleoprotein complex that may play a dual role in defense and/or virulence. In this review, we describe recently reported cases of potential involvement of CRISPR-Cas systems in bacterial stress responses in general and bacterial virulence in particular.
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.
Molecular biology; Issue 67; CRISPR/Cas; endonuclease; in vitro transcription; crRNA; Cas6
The CRISPR-Cas adaptive immunity systems that are present in most Archaea and many Bacteria function by incorporating fragments of alien genomes into specific genomic loci, transcribing the inserts and using the transcripts as guide RNAs to destroy the genome of the cognate virus or plasmid. This RNA interference-like immune response is mediated by numerous, diverse and rapidly evolving Cas (CRISPR-associated) proteins, several of which form the Cascade complex involved in the processing of CRISPR transcripts and cleavage of the target DNA. Comparative analysis of the Cas protein sequences and structures led to the classification of the CRISPR-Cas systems into three Types (I, II and III).
A detailed comparison of the available sequences and structures of Cas proteins revealed several unnoticed homologous relationships. The Repeat-Associated Mysterious Proteins (RAMPs) containing a distinct form of the RNA Recognition Motif (RRM) domain, which are major components of the CRISPR-Cas systems, were classified into three large groups, Cas5, Cas6 and Cas7. Each of these groups includes many previously uncharacterized proteins now shown to adopt the RAMP structure. Evidence is presented that large subunits contained in most of the CRISPR-Cas systems could be homologous to Cas10 proteins which contain a polymerase-like Palm domain and are predicted to be enzymatically active in Type III CRISPR-Cas systems but inactivated in Type I systems. These findings, the fact that the CRISPR polymerases, RAMPs and Cas2 all contain core RRM domains, and distinct gene arrangements in the three types of CRISPR-Cas systems together provide for a simple scenario for origin and evolution of the CRISPR-Cas machinery. Under this scenario, the CRISPR-Cas system originated in thermophilic Archaea and subsequently spread horizontally among prokaryotes.
Because of the extreme diversity of CRISPR-Cas systems, in-depth sequence and structure comparison continue to reveal unexpected homologous relationship among Cas proteins. Unification of Cas protein families previously considered unrelated provides for improvement in the classification of CRISPR-Cas systems and a reconstruction of their evolution.
Open peer review
This article was reviewed by Malcolm White (nominated by Purficacion Lopez-Garcia), Frank Eisenhaber and Igor Zhulin. For the full reviews, see the Reviewers' Comments section.
The Cmr complex is an RNA-guided effector complex that cleaves invader RNA in the prokaryotic immune response mediated by the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)-Cas system. Here we report the crystal structure of a Cmr subcomplex containing Cmr2 (Cas10) and Cmr3 subunits at 2.8 Å resolution. The structure revealed a dual Ferredoxin fold and glycine-rich loops characteristic of previously known repeat-associated mysterious proteins (RAMPs) and two unique insertion elements in Cmr3 that mediate its interaction with Cmr2. Surprisingly, while mutation of both insertion elements significantly weakened Cmr3-Cmr2 interaction, they exhibit differential effects on Cmr-mediated RNA cleavage by the Cmr complex, suggesting stabilization of Cmr2-Cmr3 interactions by other subunits. Further mutational analysis of the two conserved (but non-Cmr2-binding) glycine-rich loops of Cmr3 identified a region that is likely involved in assembly or the RNA cleavage function of the Cmr complex.
Bacterial and archaeal CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) loci capture virus and plasmid sequences and use them to recognize and eliminate these invaders. CRISPR (cr)RNAs containing the acquired sequences are incorporated into effector complexes that destroy matching invader nucleic acids. The multi-component Cmr effector complex cleaves RNA targets complementary to the crRNAs. Here we report cryo-electron microscopy reconstruction of a functional Cmr complex bound with a target RNA at ∼12Å. Pairs of the Cmr4 and Cmr5 proteins form a helical core that is asymmetrically capped on each end by distinct pairs of the four remaining subunits – Cmr2 and Cmr3 at the conserved 5′ crRNA tag sequence and Cmr1 and Cmr6 near the 3′ end of the crRNA. The shape and organization of the RNA-targeting Cmr complex is strikingly similar to the DNA-targeting Cascade complex. Our results reveal a remarkably conserved architecture among very distantly related CRISPR-Cas complexes.