CRISPR Loci Research Articles

Phenotypic and genomic analysis of isopropanol and 1,3-propanediol producer Clostridium diolis DSM 15410

Clostridium diolis DSM 15410 is a type strain of solventogenic clostridium capable of conducting isopropanol-butanol-ethanol fermentation. By studying its growth on different carbohydrates, we verified its ability to utilize glycerol and produce 1,3-propanediol and discovered its ability to produced isopropanol. Complete genome sequencing showed that its genome is a single circular chromosome and belongs to the cluster I (sensu scricto) of the genus Clostridium. By cultivation analysis we highlighted its specific behavior in comparison to two selected closely related strains. Despite the fact that several CRISPR loci were found, 16 putative prophages showed the ability to receive foreign DNA. Thus, the strain has the necessary features for future engineering of its 1,3-propanediol biosynthetic pathway and for the possible industrial utilization in the production of biofuels.

Long-run bacteria-phage coexistence dynamics under natural habitat conditions in an environmental biotechnology system.

Bacterial viruses are widespread and abundant across natural and engineered habitats. They influence ecosystem functioning through interactions with their hosts. Laboratory studies of phage-host pairs have advanced our understanding of phenotypic and genetic diversification in bacteria and phages. However, the dynamics of phage-host interactions have been seldom recorded in complex natural environments. We conducted an observational metagenomic study of the dynamics of interaction between Gordonia and their phages using a three-year data series of samples collected from a full-scale wastewater treatment plant. The aim was to obtain a comprehensive picture of the coevolution dynamics in naturally evolving populations at relatively high time resolution. Coevolution was followed by monitoring changes over time in the CRISPR loci of Gordonia metagenome-assembled genome, and reciprocal changes in the viral genome. Genome-wide analysis indicated low strain variability of Gordonia, and almost clonal conservation of the trailer end of the CRISPR loci. Incorporation of newer spacers gave rise to multiple coexisting bacterial populations. The host population carrying a shorter CRISPR locus that contain only ancestral spacers, which has not acquired newer spacers against the coexisting phages, accounted for more than half of the total host abundance in the majority of samples. Phages genome co-evolved by introducing directional changes, with no preference for mutations within the protospacer and PAM regions. Metagenomic reconstruction of time-resolved variants of host and viral genomes revealed how the complexity at the population level has important consequences for bacteria-phage coexistence.

Adaptation induced by self-targeting in a type I-B CRISPR-Cas system

Haloferax volcanii is, to our knowledge, the only prokaryote known to tolerate CRISPR-Cas–mediated damage to its genome in the WT background; the resulting cleavage of the genome is repaired by homologous recombination restoring the WT version. In mutant Haloferax strains with enhanced self-targeting, cell fitness decreases and microhomology-mediated end joining becomes active, generating deletions in the targeted gene. Here we use self-targeting to investigate adaptation in H. volcanii CRISPR-Cas type I-B. We show that self-targeting and genome breakage events that are induced by self-targeting, such as those catalyzed by active transposases, can generate DNA fragments that are used by the CRISPR-Cas adaptation machinery for integration into the CRISPR loci. Low cellular concentrations of self-targeting crRNAs resulted in acquisition of large numbers of spacers originating from the entire genomic DNA. In contrast, high concentrations of self-targeting crRNAs resulted in lower acquisition that was mostly centered on the targeting site. Furthermore, we observed naïve spacer acquisition at a low level in WT Haloferax cells and with higher efficiency upon overexpression of the Cas proteins Cas1, Cas2, and Cas4. Taken together, these findings indicate that naïve adaptation is a regulated process in H. volcanii that operates at low basal levels and is induced by DNA breaks.

Heterogeneity of Molecular Characteristics among Staphylococcus argenteus Clinical Isolates (ST2250, ST2793, ST1223, and ST2198) in Northern Taiwan.

Staphylococcus argenteus is an emerging pathogen that is recognized as non-pigmented Staphylococcus aureus. However, the molecular characteristics of S. argenteus and its virulence factors have not been well studied. The present study analyzed 96 isolates of S. argenteus recovered from blood. Identification of S. argenteus was based on results of MALDI-TOF MS and lacking crtM gene. All 96 isolates were methicillin-susceptible. Multilocus sequence typing (MLST) revealed four sequence types: ST2250 (n = 72), ST2793 (n = 12), ST1223 (n = 10), and ST2198 (n = 2). All 72 ST2250 isolates harbored CRISPR loci with polymorphism of direct repeats and spacers, but no other STs carried CRISPR loci. To date, ST2793 isolates have rarely been reported in other countries. Collagen-binding adhesin gene (cna) and staphylococcal enterotoxin type C (sec) were detected in 12 (100%) and 8 (67%) ST2793 isolates, respectively. ST1223 has been reported as food poisoning pathogens, and enterotoxin gene clusters (egc) were detected in all 10 isolates, while seb gene was detected in three isolates. Two ST2198 isolates carried bone sialoprotein-binding protein gene (bbp), belonging to agr type IV. Our focus on the heterogeneity of molecular characterization in four ST types of S. argenteus revealed that S. argenteus had been isolated as early as 2000. Each ST type of S. argenteus harbors particular genetic markers that may contribute to their virulence.

Natural CRISPR Systems and Targets in the Human Microbiome

Many bacteria resist invasive DNA by incorporating sequences into CRISPR loci, which enable sequence-specific degradation. CRISPR systems have been well-studied from isolate genomes, but culture-independent metagenomics provides a new window into their diversity. We profiled CRISPR loci and cas genes in the body-wide human microbiome using 2,355 metagenomes spanning 265 individuals, yielding functional and taxonomic profiles for 2.9 million spacers. Spacer and repeat profiles agreed qualitatively with those from isolate genomes but expanded their diversity by approximately 20-fold, with the highest spacer load present in the oral microbiome. Taxonomy of spacer carriage paralleled that of the communities in which they were contained, with functional targets enriched for viral elements but also including other microbial structural and enzymatic elements. When coupled with cas gene systems, CRISPR–Cas subtypes were highly site- and taxon-specific. Our analysis provides the first comprehensive collection of natural CRISPR–cas loci and targets in the human microbiome.

Role of Kazachstania humilis and Saccharomyces cerevisiae in the strain-specific assertiveness of Fructilactobacillus sanfranciscensis strains in rye sourdough

Sourdough is a common ingredient for baked goods as it improves their texture, shelf life and flavor. One of the dominant key species in type I sourdoughs is Fructilactobacillus sanfranciscensis (formerly Lactobacillus sanfranciscensis), which occurs with a multitude of different strains. Noticeably, this species often shares its habitat with the yeasts Saccharomyces cerevisiae or Kazachstania humilis. It is still unclear, which relationship exists between these organisms and whether it is characterized by coexistence, interaction, or mutualism. In this study, competitiveness of different F. sanfranciscensis strains in rye sourdough was examined and its dependence of co-existing yeasts was explored. In particular, it was investigated whether competitiveness of F. sanfranciscensis strains depends on the presence/absence of S. cerevisiae or K. humilis when co-inoculated in the sourdough. Competitiveness of strains was monitored in rye sourdough using the CRISPR locus length polymorphism (CLLP)—PCR for strain differentiation. It was found that F. sanfranciscensis TMW 1.1150, TMW 1.1221 and TMW 1.1597 were dominant regardless of the presence/absence of both yeast species. Dominance of F. sanfranciscensis TMW 1.392, TMW 1.907 and TMW 1.2137 was significantly and diversely influenced by the presence of S. cerevisiae or K. humilis. F. sanfranciscensis TMW 1.2138 and TMW 1.726 were not able to compete against the other F. sanfranciscensis strains. It was possible to sort the eight strains into three different groups: 1. Strain competitiveness was independent of the presence/absence of yeasts; 2. Strain competitiveness was dependent on yeast species and 3. Strains were not competitive in the presence of strains belonging to group 1 or 2. Interestingly, in fermentations that were not inoculated with any yeast a spontaneous occurrence of S. cerevisiae or K. humilis was observed depending on the synergistic competitiveness of the respective F. sanfranciscensis used. Thus, the level of competitiveness was strain specific and, in some strains, dependent on the presence/absence of specific yeast species.

CRISPR/CAS 9 Mediated Treatment for UTIs

“CRISPR" is short and used for "CRISPR-Cas9. CRISPR stands for clustered regularly interspaced short palindromic repeats. CRISPRs are specialized stretches of DNA. The protein Cas9 (or "CRISPR-associated") is an enzyme that acts like a pair of molecular scissors, capable of cutting strands of DNA and can be used in conjunction with engineered CRISPR sequences to hunt down codes and slice into them like a molecular scalpel, allowing geneticists to cut out a target gene, either to remove it or replace it with a new sequence. Therefore it is a simple and powerful tool for editing genomes to easily alter DNA sequences and amend gene function. In 1987, The CRISPR locus was first identified in Escherichia coli and discovered when a genetic structure containing 5 highly homologous repeats of 29 nucleotides separated by 32-nucleotide spacers (Ishino Y 1987).

Co-evolution within structured bacterial communities results in multiple expansion of CRISPR loci and enhanced immunity.

Type II CRISPR-Cas systems provide immunity against phages and plasmids that infect bacteria through the insertion of a short sequence from the invader's genome, known as the 'spacer', into the CRISPR locus. Spacers are transcribed into guide RNAs that direct the Cas9 nuclease to its target on the invader. In liquid cultures, most bacteria acquire a single spacer. Multiple spacer integration is a rare event which significance for immunity is poorly understood. Here, we found that when phage infections occur on solid media, a high proportion of the surviving colonies display complex morphologies that contain cells with multiple spacers. This is the result of the viral-host co-evolution, in which the immunity provided by the initial acquired spacer is easily overcome by escaper phages. Our results reveal the versatility of CRISPR-Cas immunity, which can respond with both single or multiple spacer acquisition schemes to solve challenges presented by different environments.

Sequencing and Analysis of the Genome of Propionibacterium Freudenreichii T82 Strain: Importance for Industry.

The genome of Propionibacterium freudenreichii ssp. freudenreichii T82, which has a chromosome containing 2,585,340 nucleotides with 67.3% GC content (guanine-cytosine content), is described in this paper. The total number of genes is 2308, of which 2260 are protein-coding genes and 48 are RNA genes. According to the genome analysis and the obtained results, the T82 strain can produce various compounds such as propionic acid, trehalose, glycogen, and B group vitamins (e.g., B6, B9, and B12). From protein-coding sequences (CDSs), genes related to stress adaptation, biosynthesis, metabolism, transport, secretion, and defense machinery were detected. In the genome of the T82 strain, sequences corresponding to the CRISPR loci (Clustered Regularly Interspaced Short Palindromic Repeats), antibiotic resistance, and restriction–modification system were found.

Selective loading and processing of prespacers for precise CRISPR adaptation

CRISPR-Cas immunity protects prokaryotes against invading genetic elements1. It uses the highly conserved Cas1-Cas2 complex to establish inheritable memory (spacers)2-5. How Cas1-Cas2 acquires spacers from foreign DNA fragments (prespacers) and integrates them into the CRISPR locus in the correct orientation is unclear6,7. Here, using the high spatiotemporal resolution of single-molecule fluorescence, we show that Cas1-Cas2 selects precursors of prespacers from DNA in various forms-including single-stranded DNA and partial duplexes-in a manner that depends on both the length of the DNA strand and the presence of a protospacer adjacent motif (PAM) sequence. We also identify DnaQ exonucleases as enzymes that process the Cas1-Cas2-loaded prespacer precursors into mature prespacers of a suitable size for integration. Cas1-Cas2 protects the PAM sequence from maturation, which results in the production of asymmetrically trimmed prespacers and the subsequent integration of spacers in the correct orientation. Our results demonstrate the kinetic coordination of prespacer precursor selection and PAM trimming, providing insight into the mechanisms that underlie the integration of functional spacers in the CRISPR loci.

Bacterial expression, purification, and initial characterization of a full-length Cas13b protein from Porphyromonas gingivalis

The CRISPR-Cas13b system is a recently identified Class 2, RNA-targeting CRISPR-Cas system. The system has been repurposed to achieve robust mRNA knockdown and precise RNA-editing in mammalian cells. While the CRISPR-Cas13b system has become a powerful tool for nucleic acids manipulation, the mechanisms of the system are still not fully understood. Cas13b endonucleases from different bacterial species show poor overall sequence homologies, suggesting that structural (and probably functional) diversities may exist. It is therefore important to study CRISPR-Cas13b cases from different bacterial species. Here we report the expression, purification, and initial characterization of a Cas13b endonuclease that is associated with the 8th putative CRISPR locus from Porphyromonas gingivalis genome (Pgi8Cas13b). The full-length Pgi8Cas13b protein (1119 residues) was successfully expressed in E. Coli cells, and purified by affinity and ion-exchange chromatography methods. The purified protein is biologically active, being able to bind its cognate crRNA with high specificity and affinity. Preparation of biologically active Pgi8Cas13b protein provides the basis for further in vitro biochemical and biophysical studies of the Pgi8Cas13b CRISPR system.

Casposase structure and the mechanistic link between DNA transposition and spacer acquisition by CRISPR-Cas.

Key to CRISPR-Cas adaptive immunity is maintaining an ongoing record of invading nucleic acids, a process carried out by the Cas1-Cas2 complex that integrates short segments of foreign genetic material (spacers) into the CRISPR locus. It is hypothesized that Cas1 evolved from casposases, a novel class of transposases. We show here that the Methanosarcina mazei casposase can integrate varied forms of the casposon end in vitro, and recapitulates several properties of CRISPR-Cas integrases including site-specificity. The X-ray structure of the casposase bound to DNA representing the product of integration reveals a tetramer with target DNA bound snugly between two dimers in which single-stranded casposon end binding resembles that of spacer 3'-overhangs. The differences between transposase and CRISPR-Cas integrase are largely architectural, and it appears that evolutionary change involved changes in protein-protein interactions to favor Cas2 binding over tetramerization; this in turn led to preferred integration of single spacers over two transposon ends.

The effect of phage genetic diversity on bacterial resistance evolution

CRISPR-Cas adaptive immune systems are found in bacteria and archaea and provide defence against phage by inserting phage-derived sequences into CRISPR loci on the host genome to provide sequence specific immunological memory against re-infection. Under laboratory conditions the bacterium Pseudomonas aeruginosa readily evolves the high levels of CRISPR-based immunity against clonal populations of its phage DMS3vir, which in turn causes rapid extinction of the phage. However, in nature phage populations are likely to be more genetically diverse, which could theoretically impact the frequency at which CRISPR-based immunity evolves which in turn can alter phage persistence over time. Here we experimentally test these ideas and found that a smaller proportion of infected bacterial populations evolved CRISPR-based immunity against more genetically diverse phage populations, with the majority of the population evolving a sm preventing phage adsorption and providing generalised defence against a broader range of phage genotypes. However, those cells that do evolve CRISPR-based immunity in response to infection with more genetically diverse phage acquire greater numbers of CRISPR memory sequences in order to resist a wider range of phage genotypes. Despite differences in bacterial resistance evolution, the rates of phage extinction were similar in the context of clonal and diverse phage infections suggesting selection for CRISPR-based immunity or sm-based resistance plays a relatively minor role in the ecological dynamics in this study. Collectively, these data help to understand the drivers of CRISPR-based immunity and their consequences for bacteria-phage coexistence, and, more broadly, when generalised defences will be favoured over more specific defences.

Fidelity of prespacer capture and processing is governed by the PAM-mediated interactions of Cas1-2 adaptation complex in CRISPR-Cas type I-E system

Prokaryotes deploy CRISPR-Cas-based RNA-guided adaptive immunity to fend off mobile genetic elements such as phages and plasmids. During CRISPR adaptation, which is the first stage of CRISPR immunity, the Cas1-2 integrase complex captures invader-derived prespacer DNA and specifically integrates it at the leader-repeat junction as spacers. For this integration, several variants of CRISPR-Cas systems use Cas4 as an indispensable nuclease for selectively processing the protospacer adjacent motif (PAM) containing prespacers to a defined length. Surprisingly, however, a few CRISPR-Cas systems, such as type I-E, are bereft of Cas4. Despite the absence of Cas4, how the prespacers show impeccable conservation for length and PAM selection in type I-E remains intriguing. Here, using in vivo and in vitro integration assays, deep sequencing, and exonuclease footprinting, we show that Cas1-2/I-E-via the type I-E-specific extended C-terminal tail of Cas1-displays intrinsic affinity for PAM containing prespacers of variable length in Escherichia coli Although Cas1-2/I-E does not prune the prespacers, its binding protects the prespacer boundaries from exonuclease action. This ensures the pruning of exposed ends by exonucleases to aptly sized substrates for integration into the CRISPR locus. In summary, our work reveals that in a few CRISPR-Cas variants, such as type I-E, the specificity of PAM selection resides with Cas1-2, whereas the prespacer processing is co-opted by cellular non-Cas exonucleases, thereby offsetting the need for Cas4.

A Type IV-A CRISPR-Cas System in Pseudomonas aeruginosa Mediates RNA-Guided Plasmid Interference In Vivo.

Bacteria and archaea use CRISPR-Cas adaptive immune systems to destroy complementary nucleic acids using RNAs derived from CRISPR loci. Here, we provide the first functional evidence for type IV CRISPR-Cas, demonstrating that the system from Pseudomonas aeruginosa strain PA83 mediates RNA-guided interference against a plasmid in vivo, both clearing the plasmid and inhibiting its uptake. This interference depends on the putative NTP-dependent helicase activity of Csf4/DinG.

A glance at genome editing with CRISPR-Cas9 technology.

In recent years, CRISPR-Cas9 technology is widely acknowledged for having major applications in the field of biotechnology for editing genome of any organism to treat a variety of complex diseases and for other purposes. The acronym 'CRISPR-Cas' stands for clustered regularly interspaced short palindromic repeats-CRISPR-associated genes. This genetic organization exists in prokaryotic organisms and aids in the development of adaptive immunity since a protein called Cas9 nuclease cleaves specific target nucleic acid sequences from foreign invaders and destroys them. This mode of action has gained interest of the researchers to understand the insights of CRISPR-Cas9 technology. Here, we review that CRISPR-Cas organization is restricted to two classes and possesses different protein effectors. We also review the architecture of CRISPR loci, mechanism involved in genome editing by CRISPR-Cas9 technology and pathways of repairing double-strand breaks (DSBs) generated during the process of genome editing. This review also presents the strategies to increase the Cas9 specificity and reduce off-target activity to achieve accurate genome editing. Further, this review provides information on CRISPR tools used for genome editing, databases that are required for storing data on loci, strategies for delivering CRISPR-Cas9 to cells under study and applications of CRISPR-Cas9 to various fields. Safety measures are implemented on this technology to avoid misuse or ethical issues. We also discuss about the future aspects and potential applications of CRISPR-Cas9 technology required mainly for the treatment of dreadful diseases, crop improvement as well as genetic improvement in human.

Bacterial biodiversity drives the evolution of CRISPR-based phage resistance.

Approximately half of all bacterial species encode CRISPR-Cas adaptive immune systems1, which provide immunological memory by inserting short DNA sequences from phage and other parasitic DNA elements into CRISPR loci on the host genome2. Whereas CRISPR loci evolve rapidly in natural environments3,4, bacterial species typically evolve phage resistance by the mutation or loss of phage receptors under laboratory conditions5,6. Here, we report how this discrepancy may in part be explained by differences in the biotic complexity of in vitro and natural environments7,8. Specifically, using the opportunistic pathogen Pseudomonas aeruginosa and its phage DMS3vir, we show that coexistence with other human pathogens amplifies the fitness trade-offs associated with phage receptor mutation, and therefore tips the balance in favour of CRISPR-based resistance evolution. We also demonstrate that this has important knock-on effects for P. aeruginosa virulence, which became attenuated only if the bacteria evolved surface-based resistance. Our data reveal that the biotic complexity of microbial communities in natural environments is an important driver of the evolution of CRISPR-Cas adaptive immunity, with key implications for bacterial fitness and virulence.

High-throughput screen reveals sRNAs regulating crRNA biogenesis by targeting CRISPR leader to repress Rho termination

Discovery of CRISPR-Cas systems is one of paramount importance in the field of microbiology. Currently, how CRISPR-Cas systems are finely regulated remains to be defined. Here we use small regulatory RNA (sRNA) library to screen sRNAs targeting type I-F CRISPR-Cas system through proximity ligation by T4 RNA ligase and find 34 sRNAs linking to CRISPR loci. Among 34 sRNAs for potential regulators of CRISPR, sRNA pant463 and PhrS enhance CRISPR loci transcription, while pant391 represses their transcription. We identify PhrS as a regulator of CRISPR-Cas by binding CRISPR leaders to suppress Rho-dependent transcription termination. PhrS-mediated anti-termination facilitates CRISPR locus transcription to generate CRISPR RNA (crRNA) and subsequently promotes CRISPR-Cas adaptive immunity against bacteriophage invasion. Furthermore, this also exists in type I-C/-E CRISPR-Cas, suggesting general regulatory mechanisms in bacteria kingdom. Our findings identify sRNAs as important regulators of CRISPR-Cas, extending roles of sRNAs in controlling bacterial physiology by promoting CRISPR-Cas adaptation priming.

CRISPR DNA elements controlling site-specific spacer integration and proper repeat length by a Type II CRISPR-Cas system.

CRISPR–Cas systems provide heritable immunity against viruses by capturing short invader DNA sequences, termed spacers, and incorporating them into the CRISPR loci of the prokaryotic host genome. Here, we investigate DNA elements that control accurate spacer uptake in the type II-A CRISPR locus of Streptococcus thermophilus. We determined that purified Cas1 and Cas2 proteins catalyze spacer integration with high specificity for CRISPR repeat junctions. We show that 10 bp of the CRISPR leader sequence is critical for stimulating polarized integration preferentially at the repeat proximal to the leader. Spacer integration proceeds through a two-step transesterification reaction where the 3′ hydroxyl groups of the spacer target both repeat borders on opposite strands. The leader-proximal end of the repeat is preferentially targeted for the first site of integration through recognition of sequences spanning the leader-repeat junction. Subsequently, second-site integration at the leader-distal end of the repeat is specified by multiple determinants including a length-defining mechanism relying on a repeat element proximal to the second site of integration. Our results highlight the intrinsic ability of type II Cas1/Cas2 proteins to coordinate directional and site-specific spacer integration into the CRISPR locus to ensure precise duplication of the repeat required for CRISPR immunity.

Analysis of the structures of confirmed and questionable CRISPR loci in 325 Staphylococcus genomes.

The CRISPR-Cas (clustered regular interspaced short palindromic repeats and CRISPR-associated proteins) system is a newly discovered immune defense system in the genome of prokaryotes, which can resist the invasion of foreign genetic elements, such as plasmids or phage. In this study, 154 strains of Staphylococcus published in the CRISPRDatabase and 171 strains included in NCBI were downloaded, the confirmed and questionable CRISPR loci of which were analyzed by bioinformatics methods, including their distribution, characteristics of the structure (including the direct repeats, spacers and cas genes), and the relationship between the presence of CRISPR and the mecA gene. Meanwhile, a comprehensive analysis of orphan CRISPR arrays was performed on this basis. A total of 196 confirmed and 1757 questionable CRISPR loci were found in 325 Staphylococcus genomes. Only 25 strains contained cas genes, which were classified into III-A (48.1%) and II-C (51.9%). The difference between the presence of the cas gene and the carrying rate of mecA was statistically significant, and they were negatively correlated. A total of 137 confirmed and 1755 questionable CRISPR loci were assumed to be false-CRISPR. The present study also analyzed the questionable CRISPR array for the first time while analyzing the confirmed CRISPR array in the Staphylococcal genome and screened the false-CRISPR elements in the orphan CRISPR array.