Explore the vast range of titles available.

Key Points CRISPR–Cas (clustered regularly interspaced short palindromic repeats–CRISPR-associated proteins) is an adaptive immune system in bacteria and archaea, which is categorized into three distinct types (known as type I, type II and type III) that differ in their compositions and mechanisms of action. Adaptive immunity occurs in three distinct stages: acquisition, expression and interference. In all three types of CRISPR–Cas system, spacer acquisition relies on a pair of conserved Cas proteins (Cas1 and Cas2), which are probably assisted by a variable set of additional (potentially both Cas and non-Cas) enzymes. Primary processing of CRISPR RNAs (crRNAs) is catalysed either by Cas6-like ribonucleases (in the type I and type III systems) or by RNase III (which targets the crRNA and transactivating crRNA (tracrRNA) duplex in type II systems). Mature crRNAs form CRISPR ribonucleoprotein (crRNP) complexes by associating with either Cascade-like multiprotein complexes (in type I and type III systems) or the multidomain Cas9 protein (in type II systems). Discrimination of 'self' nucleic acid from 'non-self' nucleic acid enables crRNP complexes to specifically target invading nucleic acid (usually DNA). Some complexes recruit additional nucleases (for example, Cas3 in type I systems), whereas other crRNPs have intrinsic nuclease domains (for example, Cas9 in type II systems). Fundamental studies have elucidated many mechanistic features of CRISPR–Cas functionality by integrating genetics, biochemistry and structural biology. This has provided an excellent basis for developing a wide range of applications, from the manipulation of gene expression in bacteria to genome editing in eukaryotes. In this Review, van der Oost et al . summarize the recent structural and biochemical insights into the molecular mechanisms of RNA-guided interference by CRISPR–Cas systems in bacteria and archaea. By comparing the three main types of CRISPR–Cas systems, they highlight the unique and conserved properties of the system and also discuss outstanding questions that require further study. Bacteria and archaea have evolved sophisticated adaptive immune systems, known as CRISPR–Cas (clustered regularly interspaced short palindromic repeats–CRISPR-associated proteins) systems, which target and inactivate invading viruses and plasmids. Immunity is acquired by integrating short fragments of foreign DNA into CRISPR loci, and following transcription and processing of these loci, the CRISPR RNAs (crRNAs) guide the Cas proteins to complementary invading nucleic acid, which results in target interference. In this Review, we summarize the recent structural and biochemical insights that have been gained for the three major types of CRISPR–Cas systems, which together provide a detailed molecular understanding of the unique and conserved mechanisms of RNA-guided adaptive immunity in bacteria and archaea.

Clustered regularly interspaced short palindromic repeats (CRISPRs) are essential components of RNA-guided adaptive immune systems that protect bacteria and archaea from viruses and plasmids. In Escherichia coli, short CRISPR-derived RNAs (crRNAs) assemble into a 405-kilodalton multisubunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defense). Here we present the 3.24 angstrom resolution x-ray crystal structure of Cascade. Eleven proteins and a 61-nucleotide crRNA assemble into a seahorse-shaped architecture that binds double-stranded DNA targets complementary to the crRNA-guide sequence. Conserved sequences on the 3′ and 5′ ends of the crRNA are anchored by proteins at opposite ends of the complex, whereas the guide sequence is displayed along a helical assembly of six interwoven subunits that present five-nucleotide segments of the crRNA in pseudo–A-form configuration. The structure of Cascade suggests a mechanism for assembly and provides insights into the mechanisms of target recognition.

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.

Significance Bacteria can repel invader DNA and RNA molecules by using an adaptive immunity mechanism called clustered regularly interspaced short palindromic repeats (CRISPRs)-Cas. CRISPR loci in a host genome are a repository of DNA fragments obtained from previous encounters with an invader, which can be transcribed and activated into short RNA molecules (crRNA) with sequences complementary to invader DNA or RNA. In some CRISPR-Cas systems, crRNA is assembled into a targeting complex called “Cascade” that seeks invader DNA to form an R-loop that triggers recruitment of a nuclease-helicase, Cas3, to destroy invader DNA. In this study, we show atomic resolution structures of a full-length Cas3, revealing how Cas3 coordinates binding, ATP-dependent translocation, and nuclease digestion of invader DNA. Mobile genetic elements in bacteria are neutralized by a system based on clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins. Type I CRISPR-Cas systems use a “Cascade” ribonucleoprotein complex to guide RNA specifically to complementary sequence in invader double-stranded DNA (dsDNA), a process called “interference.” After target recognition by Cascade, formation of an R-loop triggers recruitment of a Cas3 nuclease-helicase, completing the interference process by destroying the invader dsDNA. To elucidate the molecular mechanism of CRISPR interference, we analyzed crystal structures of Cas3 from the bacterium Thermobaculum terrenum , with and without a bound ATP analog. The structures reveal a histidine-aspartate (HD)-type nuclease domain fused to superfamily-2 (SF2) helicase domains and a distinct C-terminal domain. Binding of ATP analog at the interface of the SF2 helicase RecA-like domains rearranges a motif V with implications for the enzyme mechanism. The HD-nucleolytic site contains two metal ions that are positioned at the end of a proposed nucleic acid-binding tunnel running through the SF2 helicase structure. This structural alignment suggests a mechanism for 3′ to 5′ nucleolytic processing of the displaced strand of invader DNA that is coordinated with ATP-dependent 3′ to 5′ translocation of Cas3 along DNA. In agreement with biochemical studies, the presented Cas3 structures reveal important mechanistic details on the neutralization of genetic invaders by type I CRISPR-Cas systems.

Background In recent years the emergence of multidrug resistant Klebsiella pneumoniae strains has been an increasingly common event. This opportunistic species is one of the five main bacterial pathogens that cause hospital infections worldwide and multidrug resistance has been associated with the presence of high molecular weight plasmids. Plasmids are generally acquired through horizontal transfer and therefore is possible that systems that prevent the entry of foreign genetic material are inactive or absent. One of these systems is CRISPR/Cas. However, little is known regarding the clustered regularly interspaced short palindromic repeats and their associated Cas proteins (CRISPR/Cas) system in K. pneumoniae. The adaptive immune system CRISPR/Cas has been shown to limit the entry of foreign genetic elements into bacterial organisms and in some bacteria it has been shown to be involved in regulation of virulence genes. Thus in this work we used bioinformatics tools to determine the presence or absence of CRISPR/Cas systems in available K. pneumoniae genomes. Results The complete CRISPR/Cas system was identified in two out of the eight complete K. pneumoniae genomes sequences and in four out of the 44 available draft genomes sequences. The cas genes in these strains comprises eight cas genes similar to those found in Escherichia coli , suggesting they belong to the type I-E group, although their arrangement is slightly different. As for the CRISPR sequences, the average lengths of the direct repeats and spacers were 29 and 33 bp, respectively. BLAST searches demonstrated that 38 of the 116 spacer sequences (33%) are significantly similar to either plasmid, phage or genome sequences, while the remaining 78 sequences (67%) showed no significant similarity to other sequences. The region where the CRISPR/Cas systems were located is the same in all the Klebsiella genomes containing it, it has a syntenic architecture, and is located among genes encoding for proteins likely involved in metabolism and resistance to antibiotics. Conclusions The CRISPR/Cas system is not widely distributed in K. pneumoniae genomes, those present most likely belong to type I-E with few differences from the arrangement of the cse3 gene and most of the spacers have not been are not described yet. Given that the CRISPR/Cas system is scarcely distributed among K. pneumoniae genomes it is not clear whether it is involved in either immunity against foreign genetic material or virulence. We consider that this study represents a first step to understand the role of CRISPR/Cas in K. pneumoniae .

Developing a new strategy for an efficient targeted genome editing has always been a great perspective in biology. Although different approaches have been suggested in the last three decades, each one is confronting with limitations. CRISPR-Cas complex is a bacterial-derived system which made a breakthrough in the area of genome editing. This paper presents a brief history of CRISPR genome editing and discusses thoroughly how it works in bacteria and mammalians. At the end, some applications and challenges of this growing research area are also reviewed. In addition to moving the boundaries of genetics, CRISPR-Cas can also provide the ground for fundamental advances in other fields of biological sciences.

Nodule bacteria (rhizobia) forming nitrogen-fixing symbiosis with legumes have a multicomponent genome represented by chromosome and a number of plasmids of different sizes. Genes related to symbiotic activity of Sinorhizobium meliloti are located predominantly on two megaplasmids SMa and SMb (1.35 and 1.68 Mb, correspondingly). Genomes at about 80% of native rhizobia nodulating alfalfa contained cryptic plasmids with sizes varied from 7 up to 450 kb. Functional role of this class of plasmids remained unclear up to the postgenomic era. Using two high-throughput sequencing methods (MiSeq and nanopore), the complete genome sequences of three S. meliloti strains native to the Aral Sea area were done in this work. Two cryptic plasmids of the strains AK23 and AK89, and two cryptic plasmids of the strain AK555 were assembled and annotated by using bioinformatics approaches (Flye, Racon, Medaka, Pilon, Prokka). Whole genome sequence data for the strains AK21 and AK83 harboring per two cryptic plasmids are from GenBank. The objective of the study was a joint comparative analysis of the 8 cryptic plasmids of the 5 native strains of S. meliloti from one population adapted to saline environment. The average size of three and two plasmids was at about 225 and 400 kb, correspondingly, while sizes of other three plasmids was greatly different (from 31 kb up to 152 kb). Plasmids of similar size (209 kb) showed 99% identity in genetically distinct strains, besides that replicon of SMd (31 kb) of the AK555 showed 86% homology to half-replicon of pSINME02 of the AK83. All plasmids were screened for replication systems and repABC locus was detected in cases of the 6 plasmids, while some of these and other two plasmids contained additional locus, or single repA, repB or repC genes. No correlation between sizes and replication systems was detected for tested plasmids. The sequences of all plasmids were analyzed for ORFs and their affiliation with COGs groups. The number of ORFs on tested plasmid replicons was ranged from 34 up to 506, while in each particular case all detected ORFs were belonged to the next four functional groups: i) cellular processes and signaling, ii) information storage and processing, iii) metabolism and iv) poorly characterized. Proteins homologous to type IV restriction endonucleases responsible for protection of bacteria cells from invading foreign DNA, like phages, were identified on 209 kb plasmids. These proteins are relating to bacterial immune systems. Summarizing, cryptic plasmids of native S. meliloti strains adapted to salinized environment are varied greatly in sizes, replication systems and harbored ORFs relating to vitality and immune system of bacteria. Obtained data strongly evident that nonsymbiotic plasmids of S. meliloti involving in horizontal gene transfer harbored ORFs essential for environmental fitness of rhizobia.

The intestinal microbiome is emerging as a critical factor in health and disease. The microbes, although spatially restricted to the gut, are communicating and modulating the function of distant organs such as the brain. Stroke and other neurological disorders are associated with a disrupted microbiota. In turn, stroke-induced dysbiosis has a major impact on the disease outcome by modulating the immune response. In this review, we present current knowledge on the role of the gut microbiome in stroke, one of the most devastating brain disorders worldwide with very limited therapeutic options, and we discuss novel insights into the gut-immune-brain axis after an ischemic insult. Understanding the nature of the gut bacteria-brain crosstalk may lead to microbiome-based therapeutic approaches that can improve patient recovery.

Background Restriction-modification (R-M) systems are rudimentary bacterial immune systems. The main components include restriction enzyme (R), which cuts specific unmethylated DNA sequences, and the methyltransferase (M), which protects the same DNA sequences. The expression of R-M system components is considered to be tightly regulated, to ensure successful establishment in a naïve bacterial host. R-M systems are organized in different architectures (convergent or divergent) and are characterized by different features, i.e. binding cooperativities, dissociation constants of dimerization, translation rates, which ensure this tight regulation. It has been proposed that R-M systems should exhibit certain dynamical properties during the system establishment, such as: i ) a delayed expression of R with respect to M, ii ) fast transition of R from “OFF” to “ON” state, iii ) increased stability of the toxic molecule (R) steady-state levels. It is however unclear how different R-M system features and architectures ensure these dynamical properties, particularly since it is hard to address this question experimentally. Results To understand design of different R-M systems, we computationally analyze two R-M systems, representative of the subset controlled by small regulators called ‘C proteins’, and differing in having convergent or divergent promoter architecture. We show that, in the convergent system, abolishing any of the characteristic system features adversely affects the dynamical properties outlined above. Moreover, an extreme binding cooperativity, accompanied by a very high dissociation constant of dimerization, observed in the convergent system, but absent from other R-M systems, can be explained in terms of the same properties. Furthermore, we develop the first theoretical model for dynamics of a divergent R-M system, which does not share any of the convergent system features, but has overlapping promoters. We show that i ) the system dynamics exhibits the same three dynamical properties, ii ) introducing any of the convergent system features to the divergent system actually diminishes these properties. Conclusions Our results suggest that different R-M architectures and features may be understood in terms of constraints imposed by few simple dynamical properties of the system, providing a unifying framework for understanding these seemingly diverse systems. We also provided predictions for the perturbed R-M systems dynamics, which may in future be tested through increasingly available experimental techniques, such as re-engineering R-M systems and single-cell experiments.

Bacteriophage contamination remains a persistent and costly challenge in industrial bio-manufacturing. Traditional control strategies rely heavily on physical exclusion and chemical disinfection, yet these passive measures often fail to address the rapid evolutionary adaptation of phages and their persistence in complex fermentation environments. Recent genomic and biochemical discoveries have revealed a diverse arsenal of bacterial antiviral immune systems beyond the classical Restriction-Modification and CRISPR-Cas pathways, including cyclic oligonucleotide-based signaling systems and various abortive infection mechanisms. This review systematically summarizes the latest advances in bacterial anti-phage defense mechanisms, categorizing them into adsorption inhibition, replication interference, nucleic acid degradation, and population-level suicide defense. Furthermore, we discuss the application of synthetic biology in integrating these defense modules to construct broad-spectrum “pan-immune” microbial chassis. This active defense strategy offers a fundamental solution to phage predation and provides a theoretical basis for developing robust next-generation cell factories.