Clustered Regularly Interspaced Short Palindromic Repeats Cas Research Articles

The subtype I-F CRISPR–Cas system influences pathogenicity island retention in Pectobacterium atrosepticum via crRNA generation and Csy complex formation

CRISPR (clustered regularly interspaced short palindromic repeats) arrays and Cas (CRISPR-associated) proteins confer acquired resistance against mobile genetic elements in a wide range of bacteria and archaea. The phytopathogen Pectobacterium atrosepticum SCRI1043 encodes a single subtype I-F CRISPR system, which is composed of three CRISPR arrays and the cas operon encoding Cas1, Cas3 (a Cas2-Cas3 fusion), Csy1, Csy2, Csy3 and Cas6f (Csy4). The CRISPR arrays are transcribed into pre-crRNA (CRISPR RNA) and then processed by Cas6f to generate crRNAs. Furthermore, the formation of Cas protein complexes has been implicated in both the interference and acquisition stages of defence. In the present paper, we discuss the development of tightly controlled 'programmable' CRISPR arrays as tools to investigate CRISPR-Cas function and the effects of chromosomal targeting. Finally, we address how chromosomal targeting by CRISPR-Cas can cause large-scale genome deletions, which can ultimately influence bacterial evolution and pathogenicity.

The RNA- and DNA-targeting CRISPR–Cas immune systems of Pyrococcus furiosus

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.

Genomic impact of CRISPR immunization against bacteriophages

CRISPR (clustered regularly interspaced short palindromic repeats) together with CAS (RISPR-associated) genes form the CRISPR-Cas immune system, which provides sequence-specific adaptive immunity against foreign genetic elements in bacteria and archaea. Immunity is acquired by the integration of short stretches of invasive DNA as novel 'spacers' into CRISPR loci. Subsequently, these immune markers are transcribed and generate small non-coding interfering RNAs that specifically guide nucleases for sequence-specific cleavage of complementary sequences. Among the four CRISPR-Cas systems present in Streptococcus thermophilus, CRISPR1 and CRISPR3 have the ability to readily acquire new spacers following bacteriophage or plasmid exposure. In order to investigate the impact of building CRISPR-encoded immunity on the host chromosome, we determined the genome sequence of a BIM (bacteriophage-insensitive mutant) derived from the DGCC7710 model organism, after four consecutive rounds of bacteriophage challenge. As expected, active CRISPR loci evolved via polarized addition of several novel spacers following exposure to bacteriophages. Although analysis of the draft genome sequence revealed a variety of SNPs (single nucleotide polymorphisms) and INDELs (insertions/deletions), most of the in silico differences were not validated by Sanger re-sequencing. In addition, two SNPs and two small INDELs were identified and tracked in the intermediate variants. Overall, building CRISPR-encoded immunity does not significantly affect the genome, which allows the maintenance of important functional properties in isogenic CRISPR mutants. This is critical for the development and formulation of sustainable and robust next-generation starter cultures with increased industrial lifespans.

Are bacteriophage defence and virulence two sides of the same coin in Campylobacter jejuni?

The continuous battle for survival in the environment has led to the development or acquisition of sophisticated defence systems in bacteria. These defence systems have contributed to the survival of the bacterial species in the environment for millions of years. Some systems appear to have evolved in a number of pathogenic bacteria towards a role in virulence and host immune evasion. Recently, different bacterial cell envelope components from diverse bacterial species have been linked not only to bacteriophage defence, but also to virulence features. In the present review we focus specifically on the bacterial cell envelope-expressed sialic-acid-containing LOS (lipo-oligosaccharide) structures and Type II CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) genes that both occur in specific Gram-negative pathogens. In Campylobacter jejuni circumstantial evidence points at a potential intertwined dual function between sialylated LOS structures and subtype II-C CRISPR-Cas, i.e. in phage defence and virulence. In the present review we discuss whether a dual functionality of sialylated LOS and subtype II-C CRISPR-Cas is exclusive to C. jejuni only or could be more widespread within the group of Type II CRISPR-Cas-harbouring bacteria. We conclude from the literature that, at least in C. jejuni, circumstantial evidence exists for a complex intertwined dual functionality between sialylated LOS and Type II CRISPR-Cas, and that other bacteria show similar genomic signatures.

Requirements for a successful defence reaction by the CRISPR–Cas subtype I-B system

Uptake of foreign mobile genetic elements is often detrimental and can result in cell death. For protection against invasion, prokaryotes have developed several defence mechanisms, which take effect at all stages of infection; an example is the recently discovered CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) immune system. This defence system directly degrades invading genetic material and is present in almost all archaea and many bacteria. Current data indicate a large variety of mechanistic molecular approaches. Although almost all archaea carry this defence weapon, only a few archaeal systems have been fully characterized. In the present paper, we summarize the prerequisites for the detection and degradation of invaders in the halophilic archaeon Haloferax volcanii. H. volcanii encodes a subtype I-B CRISPR-Cas system and the defence can be triggered by a plasmid-based invader. Six different target-interference motifs are recognized by the Haloferax defence and a 9-nt non-contiguous seed sequence is essential. The repeat sequence has the potential to fold into a minimal stem-loop structure, which is conserved in haloarchaea and might be recognized by the Cas6 endoribonuclease during the processing of CRISPR loci into mature crRNA (CRISPR RNA). Individual crRNA species were present in very different concentrations according to an RNA-Seq analysis and many were unable to trigger a successful defence reaction. Recognition of the plasmid invader does not depend on its copy number, but instead results indicate a dependency on the type of origin present on the plasmid.

The basic building blocks and evolution of CRISPR–Cas systems

CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) is an adaptive immunity system in bacteria and archaea that functions via a distinct self/non-self recognition mechanism that involves unique spacers homologous with viral or plasmid DNA and integrated into the CRISPR loci. Most of the Cas proteins evolve under relaxed purifying selection and some underwent dramatic structural rearrangements during evolution. In many cases, CRISPR-Cas system components are replaced either by homologous or by analogous proteins or domains in some bacterial and archaeal lineages. However, recent advances in comparative sequence analysis, structural studies and experimental data suggest that, despite this remarkable evolutionary plasticity, all CRISPR-Cas systems employ the same architectural and functional principles, and given the conservation of the principal building blocks, share a common ancestry. We review recent advances in the understanding of the evolution and organization of CRISPR-Cas systems. Among other developments, we describe for the first time a group of archaeal cas1 gene homologues that are not associated with CRISPR-Cas loci and are predicted to be involved in functions other than adaptive immunity.

Hot and crispy: CRISPR–Cas systems in the hyperthermophile Sulfolobus solfataricus

The CRISPR (clustered regularly interspaced short palindromic repeats) and Cas (CRISPR-associated) genes are widely spread in bacteria and archaea, representing an intracellular defence system against invading viruses and plasmids. In the system, fragments from foreign DNA are captured and integrated into the host genome at the CRISPR locus. The locus is transcribed and the resulting RNAs are processed by Cas6 into small crRNAs (CRISPR RNAs) that guide a variety of effector complexes to degrade the invading genetic elements. Many bacteria and archaea have one major type of effector complex. However, Sulfolobus solfataricus strain P2 has six CRISPR loci with two families of repeats, four cas6 genes and three different types of effector complex. These features make S. solfataricus an important model for studying CRISPR-Cas systems. In the present article, we review our current understanding of crRNA biogenesis and its effector complexes, subtype I-A and subtype III-B, in S. solfataricus. We also discuss the differences in terms of mechanisms between the subtype III-B systems in S. solfataricus and Pyrococcus furiosus.

Cas9-dependent endogenous gene regulation is required for bacterial virulence

CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems are known to mediate bacterial defence against foreign nucleic acids. We recently demonstrated a non-canonical role for a CRISPR-Cas system in controlling endogenous gene expression, which had not previously been appreciated. In the present article, we describe the studies that led to this discovery, beginning with an unbiased genome-wide screen to identify virulence genes in the intracellular pathogen Francisella novicida. A gene annotated as encoding a hypothetical protein, but which we now know encodes the Cas protein Cas9, was identified as one of the most critical to the ability of F. novicida to replicate and survive during murine infection. Subsequent studies revealed a role for this protein in evasion of the host innate immune response. Specifically, Cas9 represses the expression of a BLP (bacterial lipoprotein) that could otherwise be recognized by TLR2 (Toll-like receptor 2), a host protein involved in initiating an antibacterial pro-inflammatory response. By repressing BLP levels, Cas9 mediates evasion of TLR2, promoting bacterial virulence. Finally, we described the molecular mechanism by which Cas9 functions in complex with two small RNAs to target the mRNA encoding the BLP for degradation. This work greatly broadened the paradigm for CRISPR-Cas function, highlighting a role in gene regulation that could be conserved in numerous bacteria, and elucidating its integral contribution to bacterial pathogenesis.

RNA-Seq analyses reveal CRISPR RNA processing and regulation patterns

In bacteria and archaea, RNA-Seq deep sequencing methodology allows for the detection of abundance and processing sites of the small RNAs that comprise a CRISPR (clustered regularly interspaced short palindromic repeats) RNome. Comparative analyses of these CRISPR RNome sets highlight conserved patterns that include the gradual decline of CRISPR RNA abundance from the leader-proximal to the leader-distal end. In the present review, we discuss exceptions to these patterns that indicate the extensive impact of individual spacer sequences on CRISPR array transcription and RNA maturation. Spacer sequences can contain promoter and terminator elements and can promote the formation of CRISPR RNA-anti-CRISPR RNA duplexes. In addition, potential RNA duplex formation with host tRNA was observed. These factors can influence the functionality of CRISPR-Cas (CRISPR-associated) systems and need to be considered in the design of synthetic CRISPR arrays.

CRISPR adaptation in Escherichia coli subtypeI-E system

The CRISPRs (clustered regularly interspaced short palindromic repeats) and their associated Cas (CRISPR-associated) proteins are a prokaryotic adaptive defence system against foreign nucleic acids. The CRISPR array comprises short repeats flanking short segments, called 'spacers', which are derived from foreign nucleic acids. The process of spacer insertion into the CRISPR array is termed 'adaptation'. Adaptation allows the system to rapidly evolve against emerging threats. In the present article, we review the most recent studies on the adaptation process, and focus primarily on the subtypeI-E CRISPR-Cas system of Escherichia coli.

Zinc Fingers, TAL Effectors, or Cas9-Based DNA Binding Proteins: What’s Best for Targeting Desired Genome Loci?

Programmable DNA binding proteins (PDPs) that selectively bind user-defined nucleotide sequences can navigate protein domains to any desired target within the genome. Different functional domains can be fused to PDPs to achieve (1) activation or repression of transcription, (2) nicking or cleavage of DNA, or (3) modification or removal of epigenetic marks at the targeted genome spot. Given their broad applicability, the development of highly specific, easy-to-use PDPs has been a holy grail of biotechnology since the determination of the 3-D structure of zinc fingers (ZFs) bound to DNA in 1991 (Pavletich and Pabo, 1991).

Molecular mechanisms of CRISPR-mediated microbial immunity

Bacteriophages (phages) infect bacteria in order to replicate and burst out of the host, killing the cell, when reproduction is completed. Thus, from a bacterial perspective, phages pose a persistent lethal threat to bacterial populations. Not surprisingly, bacteria evolved multiple defense barriers to interfere with nearly every step of phage life cycles. Phages respond to this selection pressure by counter-evolving their genomes to evade bacterial resistance. The antagonistic interaction between bacteria and rapidly diversifying viruses promotes the evolution and dissemination of bacteriophage-resistance mechanisms in bacteria. Recently, an adaptive microbial immune system, named clustered regularly interspaced short palindromic repeats (CRISPR) and which provides acquired immunity against viruses and plasmids, has been identified. Unlike the restriction–modification anti-phage barrier that subjects to cleavage any foreign DNA lacking a protective methyl-tag in the target site, the CRISPR–Cas systems are invader-specific, adaptive, and heritable. In this review, we focus on the molecular mechanisms of interference/immunity provided by different CRISPR–Cas systems.Electronic supplementary materialThe online version of this article (doi:10.1007/s00018-013-1438-6) contains supplementary material, which is available to authorized users.

Crystallization and preliminary X-ray diffraction analysis of the Cmr2–Cmr3 subcomplex in the CRISPR–Cas RNA-silencing effector complex

Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci, found in prokaryotes, are transcribed to produce CRISPR RNAs (crRNAs). The Cmr proteins (Cmr1-6) and crRNA form a ribonucleoprotein complex that degrades target RNAs derived from invading genetic elements. Cmr2dHD, a Cmr2 variant lacking the N-terminal putative HD nuclease domain, and Cmr3 were co-expressed in Escherichia coli cells and co-purified as a complex. The Cmr2dHD-Cmr3 complex was co-crystallized with 3'-AMP by the vapour-diffusion method. The crystals diffracted to 2.6 Å resolution using synchrotron radiation at the Photon Factory. The crystals belonged to the orthorhombic space group I222, with unit-cell parameters a = 103.9, b = 136.7, c = 192.0 Å. The asymmetric unit of the crystals is expected to contain one Cmr2dHD-Cmr3 complex with a Matthews coefficient of 3.0 Å(3) Da(-1) and a solvent content of 59%.

The Population and Evolutionary Dynamics of Phage and Bacteria with CRISPR–Mediated Immunity

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), together with associated genes (cas), form the CRISPR–cas adaptive immune system, which can provide resistance to viruses and plasmids in bacteria and archaea. Here, we use mathematical models, population dynamic experiments, and DNA sequence analyses to investigate the host–phage interactions in a model CRISPR–cas system, Streptococcus thermophilus DGCC7710 and its virulent phage 2972. At the molecular level, the bacteriophage-immune mutant bacteria (BIMs) and CRISPR–escape mutant phage (CEMs) obtained in this study are consistent with those anticipated from an iterative model of this adaptive immune system: resistance by the addition of novel spacers and phage evasion of resistance by mutation in matching sequences or flanking motifs. While CRISPR BIMs were readily isolated and CEMs generated at high rates (frequencies in excess of 10−6), our population studies indicate that there is more to the dynamics of phage–host interactions and the establishment of a BIM–CEM arms race than predicted from existing assumptions about phage infection and CRISPR–cas immunity. Among the unanticipated observations are: (i) the invasion of phage into populations of BIMs resistant by the acquisition of one (but not two) spacers, (ii) the survival of sensitive bacteria despite the presence of high densities of phage, and (iii) the maintenance of phage-limited communities due to the failure of even two-spacer BIMs to become established in populations with wild-type bacteria and phage. We attribute (i) to incomplete resistance of single-spacer BIMs. Based on the results of additional modeling and experiments, we postulate that (ii) and (iii) can be attributed to the phage infection-associated production of enzymes or other compounds that induce phenotypic phage resistance in sensitive bacteria and kill resistant BIMs. We present evidence in support of these hypotheses and discuss the implications of these results for the ecology and (co)evolution of bacteria and phage.

Conservation and Variability in the Structure and Function of the Cas5d Endoribonuclease in the CRISPR-Mediated Microbial Immune System

Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins form an RNA-mediated microbial immune system against invading foreign genetic elements. Cas5 proteins constitute one of the most prevalent Cas protein families in CRISPR–Cas systems and are predicted to have RNA recognition motif (RRM) domains. Cas5d is a subtype I-C-specific Cas5 protein that can be divided into two distinct subgroups, one of which has extra C-terminal residues while the other contains a longer insertion in the middle of its N-terminal RRM domain. Here, we report crystal structures of Cas5d from Streptococcus pyogenes and Xanthomonas oryzae, which respectively represent the two Cas5d subgroups. Despite a common domain architecture consisting of an N-terminal RRM domain and a C-terminal β-sheet domain, the structural differences between the two Cas5d proteins are highlighted by the presence of a unique extended helical region protruding from the N-terminal RRM domain of X. oryzae Cas5d. We also demonstrate that Cas5d proteins possess not only specific endoribonuclease activity for CRISPR RNAs but also nonspecific double-stranded DNA binding affinity. These findings suggest that Cas5d may play multiple roles in CRISPR-mediated immunity. Furthermore, the specific RNA processing was also observed between S. pyogenes Cas5d protein and X. oryzae CRISPR RNA and vice versa. This cross-species activity of Cas5d provides a special opportunity for elucidating conserved features of the CRISPR RNA processing event.

Strong bias in the bacterial CRISPR elements that confer immunity to phage

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems provide adaptive immunity against phage via spacer-encoded CRISPR RNAs that are complementary to invasive nucleic acids. Here, we challenge Streptococcus thermophilus with a bacteriophage, and used PCR-based metagenomics to monitor phage-derived spacers daily for 15 days in two experiments. Spacers that target the host chromosome are infrequent and strongly selected against, suggesting autoimmunity is lethal. In experiments that recover over half a million spacers, we observe early dominance by a few spacer sub-populations and rapid oscillations in sub-population abundances. In two CRISPR systems and in replicate experiments, a few spacers account for the majority of spacer sequences. Nearly all phage locations targeted by the acquired spacers have a proto-spacer adjacent motif (PAM), indicating PAMs are involved in spacer acquisition. We detect a strong and reproducible bias in the phage genome locations from which spacers derive. This may reflect selection for specific spacers based on location and effectiveness.

Characterization of CRISPR RNA Biogenesis and Cas6 Cleavage-Mediated Inhibition of a Provirus in the Haloarchaeon Haloferax mediterranei

The adaptive immune system comprising CRISPR (clustered regularly interspaced short palindromic repeats) arrays and cas (CRISPR-associated) genes has been discovered in a wide range of bacteria and archaea and has recently attracted comprehensive investigations. However, the subtype I-B CRISPR-Cas system in haloarchaea has been less characterized. Here, we investigated Cas6-mediated RNA processing in Haloferax mediterranei. The Cas6 cleavage site, as well as the CRISPR transcription start site, was experimentally determined, and processing of CRISPR transcripts was detected with a progressively increasing pattern from early log to stationary phase. With genetic approaches, we discovered that the lack of Cas1, Cas3, or Cas4 unexpectedly resulted in a decrease of CRISPR transcripts, while Cas5, Cas6, and Cas7 were found to be essential in stabilizing mature CRISPR RNA (crRNA). Intriguingly, we observed a CRISPR- and Cas3-independent inhibition of a defective provirus, in which the putative Cascade (CRISPR-associated complex for antiviral defense) proteins (Cas5, Cas6, Cas7, and Cas8b) were indispensably required. A sequence carried by a proviral transcript was found to be homologous to the CRISPR repeat RNA and vulnerable to Cas6-mediated cleavage, implying a distinct interference mechanism that may account for this unusual inhibition. These results provide fundamental information for the subtype I-B CRISPR-Cas system in halophilic archaea and suggest diversified mechanisms and multiple physiological functions for the CRISPR-Cas system.

Binding and cleavage of CRISPR RNA by Cas6

The CRISPR-Cas system provides many prokaryotes with acquired resistance to viruses and other mobile genetic elements. The core components of this defense system are small, host-encoded prokaryotic silencing (psi)RNAs and Cas (CRISPR-associated) proteins. Invader-derived sequences within the psiRNAs guide Cas effector proteins to recognize and silence invader nucleic acids. Critical for CRISPR-Cas defense is processing of the psiRNAs from the primary transcripts of the host CRISPR (clustered regularly interspaced short palindromic repeat) locus. Cas6, a previously identified endoribonuclease present in a wide range of prokaryotes with the CRISPR-Cas system, binds and cleaves within the repeat sequences that separate the individual invader targeting elements in the CRISPR locus transcript. In the present study, we investigated several key aspects of the mechanism of function of Cas6 in psiRNA biogenesis. RNA footprinting reveals that Pyrococcus furiosus Cas6 binds to a 7-nt (nucleotide) sequence near the 5' end of the CRISPR RNA repeat sequence, 14 nt upstream of the Cas6 cleavage site. In addition, analysis of the cleavage activity of P. furiosus Cas6 proteins with mutations at conserved residues suggests that a triad comprised of Tyr31, His46, and Lys52 plays a critical role in catalysis, consistent with a possible general acid-base RNA cleavage mechanism for Cas6. Finally, we show that P. furiosus Cas6 remains stably associated with its cleavage products, suggesting additional roles for Cas6 in psiRNA biogenesis.

Identification and characterization of E. coli CRISPR‐cas promoters and their silencing by H‐NS

Inheritable bacterial defence systems against phage infection and foreign DNA, termed CRISPR (clustered regularly interspaced short palindromic repeats), consist of cas protein genes and repeat arrays interspaced with sequences originating from invaders. The Cas proteins together with processed small spacer-repeat transcripts (crRNAs) cause degradation of penetrated foreign DNA by unknown mechanisms. Here, we have characterized previously unidentified promoters of the Escherichia coli CRISPR arrays and cas protein genes. Transcription of precursor crRNA is directed by a promoter located within the CRISPR leader. A second promoter, directing cas gene transcription, is located upstream of the genes encoding proteins of the Cascade complex. Furthermore, we demonstrate that the DNA-binding protein H-NS is involved in silencing the CRISPR-cas promoters, resulting in cryptic Cas protein expression. Our results demonstrate an active involvement of H-NS in the induction of the CRISPR-cas system and suggest a potential link between two prokaryotic defence systems against foreign DNA.