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Aims We performed in silico analysis of CRISPRcas loci from Tenacibaculum maritimum, evaluated spoligotyping as a subtyping method and genotyped uncharacterized Turkish isolates from European sea bass by multilocus sequence typing (MLST). Methods and results Spoligotyping was performed with primers designed to allow amplification and sequencing of whole CRISPR-arrays from 23 T. maritimum isolates. Twenty-three completed/draft genomes were also downloaded from the NCBI database and analysed. MLST of Turkish isolates was achieved with a well-established 7-gene scheme. Tenacibaculum maritimum genomes carry a structurally complete but partially defective class II CRISPRcas locus due to known amino acid substitutions in encoded Cas9 proteins. Our spacer identification suggests that the host range of bacteriophage P2559Y and Vibrio phage nt-1 include T. maritimum and that the most recurrent infection recorded by isolates has been with Tenacibaculum phage PTm5. Thirty-eight isolates with this CRISPRcas locus belonged to 25 spoligotypes and to 24 sequence types by MLST, respectively. According to MLST, T. maritimum isolates from Turkey are most related to previously defined sequence types ST3, ST40 and ST41 isolates from Spain, Malta and France. Conclusions The evaluated spoligotyping offers discriminatory power comparable to MLST. Significance and impact of the study Spoligotyping has potential as a quick, easy and cheap tool for subtyping of T. maritimum isolates.

In shrimp farming, the family of proteobacteria Vibrionaceae, especially the species of the genus Vibrio, represent one of the main responsible for infections in shrimp production (Litopenaeus vannamei), generating great losses to this industry. Phagotherapy emerges as a novel alternative for the control of said infections in substitution to the use of antibiotics, thanks to the specific inhibitory activity of these viruses. However, it is necessary to take into account the presence in prokaryotes of genetic sequences called clusters of regularly interspaced short palindromic repeats (CRISPR) that act as an immune system against invasion of external mobile genetic elements such as phage or plasmids. Due to its characteristics, the CRISPR/Cas system is used as a tool for gene editing. This study presents the comparative analysis of 7 CRISPR loci found in 5 sequences of complete genomes, available in the database of NCBI/GenBank, to determine the potential use of the phage strategy in shrimp farming. The CRISPR systems corresponded to types I-E, I-F and III-D. 53 % of the spacers (75/142) presented homology with plasmids, while the remaining 47 % (67/142) showed homology with bacteriophages, mostly non-typical Vibrio infective viruses. The use of phage therapy is proposed as a treatment for infections caused by members of the family Vibrionaceae in shrimp cultures, due to the low occurrence of CRISPR systems in the species studied and the low immunity to their phages, thus ensuring greater sensitivity.

Clustered regularly interspaced short palindromic repeats (CRISPR) consists of a series of regular repeat-spacer sequences. It can not only act as a natural immune system in most prokaryotes, but also be utilized as the tool of newly developed genome modification and evolutionary researches. Streptococcus thermophilus is an important model organism for the study and application of CRISPR systems. In present study, the occurrence and diversity of CRISPR-Cas systems in the genomes of S. thermophilus were investigated including 4 new sequenced strains CS5, CS9, CS18, CS20, and other 23 strains downloaded from NCBI website. 66 CRISPR/Cas systems were identified among these 27 strains and could divided into four subsystems according to the arrangement of Cas proteins, notably I-E, II-A, II-C and III-A. Overall, 26 type II-C systems, 18 type II-A systems, 13 type III-A systems, 9 type I-E systems were identified. It was mentioned that CS20 contained two type II-C systems which had not been identified in the other 26 S. thermophilus strains. Overall, 1,080 spacers were analyzed and blasted. Sequence identity searches of spacers implied that most spacers derived from partial sequences of exogenous DNA, including various bacteriophages and plasmids. Of note, a large number of novel spacers were found in this study, indicating the unique phage environment they have undergone, especially CS20 strain. In addition, the analysis of the cas1 and cas9 genes revealed the genetic relationship among CRISPR-Cas system in these strains. Furthermore, the analysis of CRISPR spacers also indicated protospacer adjacent motif (PAM) sequences. Summary of PAM sequences could lay the foundations for the application of S. thermophilus CRISPR-Cas system. Our results suggested CS5 and CS18 can be used as model strains in the research of CRISPR-Cas system, and CS20 might have greater application potential in gene editing.

The aim of this research was to analyze and compare CRIPSR loci and cas-proteins of Yersinia pseudotuberculosis strains isolated in different territories from patients with various clinical manifestations of pseudotuberculosis.Materials and Methods. Complete genomes of Y. pseudotuberculosis IP329353 (NC_006155) and IP31758 (NC_009708) were obtained from NCBI Nucleotide Database. Strains were isolated from patients with gastroenteritis and systemic infection respectively. Search, identification, and analysis of CRISPR systems were carried out by onlinetools CRISPROne, CRISPRDetect, and CRISPRTarget.Results. Analyzed strains have CRISPR-Cas systems that include one set of cas-genes and arrays situated at the long distances from each other. We defined three CRISPR arrays in Y. pseudotuberculosis IP32953: array YP1 located near cas-genes, arrays YP2 and YP3. CRISPR-Cas system of Y. pseudotuberculosis IP31758 includes two arrays – YP1 and YP3. CRISPR systems do not share similar spacers.Conclusion. CRISPR systems of the analyzed strains differ in CRISPR loci and cas-protein structures that can be used as specific molecular marks of analyzed strains during the study of intra-species variability and evolution of Y. pseudotuberculosis.

An enigmatic uncultured member of Firmicutes, Candidatus Desulforudis audaxviator (CDA), is known by its genome retrieved from the deep gold mine in South Africa, where it formed a single-species ecosystem fuelled by hydrogen from water radiolysis. It was believed that in situ conditions CDA relied on scarce energy supply and did not divide for hundreds to thousand years. We have isolated CDA strain BYF from a 2-km-deep aquifer in Western Siberia and obtained a laboratory culture growing with a doubling time of 28.5 h. BYF uses not only H(2) but also various organic electron donors for sulfate respiration. Growth required elemental iron, and ferrous iron did not substitute for it. A complex intracellular organization included gas vesicles, internal membranes, and electron-dense structures enriched in phosphorus, iron, and calcium. Genome comparison of BYF with the South African CDA revealed minimal differences mostly related to mobile elements and prophage insertions. Two genomes harbored

Approaches for recovering and analyzing genomes belonging to novel, hitherto unexplored bacterial lineages have provided invaluable insights into the metabolic capabilities and ecological roles of yet-uncultured taxa. The phylum Acidobacteria is one of the most prevalent and ecologically successful lineages on earth yet, currently, multiple lineages within this phylum remain unexplored. Here, we utilize genomes recovered from Zodletone spring, an anaerobic sulfide and sulfur-rich spring in southwestern Oklahoma, as well as from multiple disparate soil and non-soil habitats, to examine the metabolic capabilities and ecological role of members of the family UBA6911 (group18) Acidobacteria. The analyzed genomes clustered into five distinct genera, with genera Gp18_AA60 and QHZH01 recovered from soils, genus Ga0209509 from anaerobic digestors, and genera Ga0212092 and UBA6911 from freshwater habitats. All genomes analyzed suggested that members of Acidobacteria group 18 are metabolically versatile heterotrophs capable of utilizing a wide range of proteins, amino acids, and sugars as carbon sources, possess respiratory and fermentative capacities, and display few auxotrophies. Soil-dwelling genera were characterized by larger genome sizes, higher number of CRISPR loci, an expanded carbohydrate active enzyme (CAZyme) machinery enabling de-branching of specific sugars from polymers, possession of a C1 (methanol and methylamine) degradation machinery, and a sole dependence on aerobic respiration. In contrast, non-soil genomes encoded a more versatile respiratory capacity for oxygen, nitrite, sulfate, trimethylamine N-oxide (TMAO) respiration, as well as the potential for utilizing the Wood Ljungdahl (WL) pathway as an electron sink during heterotrophic growth. Our results not only expand our knowledge of the metabolism of a yet-uncultured bacterial lineage, but also provide interesting clues on how terrestrialization and niche adaptation drives metabolic specialization within the Acidobacteria.ImportanceMembers of the Acidobacteria are important players in global biogeochemical cycles, especially in soils. A wide range of Acidobacterial lineages remain currently unexplored. We present a detailed genomic characterization of genomes belonging to the family UBA6911 (also known as group 18) within the phylum Acidobacteria. The genomes belong to different genera and were obtained from soil (genera Gp18_AA60 and QHZH01), freshwater habitats (genera Ga0212092 and UBA6911), and anaerobic digestor (Genus Ga0209509). While all members of the family shared common metabolic features, e.g. heterotrophic respiratory abilities, broad substrate utilization capacities, and few auxotrophies; distinct differences between soil and non-soil genera were observed. Soil genera were characterized by expanded genomes, higher numbers of CRISPR loci, larger carbohydrate active enzyme (CAZyme) repertoire enabling monomer extractions from polymer side chains, and methylotrophic (methanol and methylamine) degradation capacities. In contrast, non-soil genera encoded more versatile respiratory capacities for utilizing nitrite, sulfate, TMAO, and the WL pathway, in addition to oxygen as electron acceptors. Our results not only broaden our understanding of the metabolic capacities within the Acidobacteria, but also, provide interesting clues on how terrestrialization shaped Acidobacteria evolution and niche adaptation.

Tetragenococcus halophilus – a halophilic lactic acid bacterium – is frequently used as a starter culture for manufacturing fermented foods. Tetragenococcus is sometimes infected with bacteriophages during fermentation for soy sauce production; however, bacteriophage infection in starter bacteria is one of the major causes of fermentation failure. Here, we obtained whole-genome sequences of the four T. halophilus strains YA5, YA163, YG2, and WJ7 and compared them with 18 previously reported genomes. We elucidated five types of clustered regularly interspaced short palindromic repeat (CRISPR) loci in seven genomes using comparative genomics with a particular focus on CRISPR elements. CRISPR1 was conserved in the four closely related strains 11, YA5, YA163, and YG2, and the spacer sequences were partially retained in each strain, suggesting that partial deletions and accumulation of spacer sequences had occurred independently after divergence of each strain. The host range for typical bacteriophages is narrow and strain-specific thus these accumulation/deletion events may be responsible for differences in resistance to bacteriophages between bacterial strains. Three CRISPR elements, CRISPR1 in strains 11, YA5, YA163, and YG2, CRISPR2 in strain WJ7, and CRISPR2 in strain MJ4, were inserted in almost the same genomic regions, indicating that several independent insertions had occurred in this region. As these elements belong to class 1 type I-C CRISPR group, the results suggested that this site is a hotspot for class 1, type I-C CRISPR loci insertion. Thus, T. halophilus genomes may have acquired strain-specific bacteriophage-resistance through repeated insertion of CRISPR loci and accumulation/deletion events of their spacer sequences.

Some prokaryotes possess CRISPR-Cas systems that provide adaptive immunity to viruses guided by DNA segments called spacers acquired from invading phage. However, the patchy incidence and limited memory breadth of CRISPR-Cas systems suggest that their fitness benefits are offset by costs. Here, we propose that cross-reactive CRISPR targeting can lead to heterologous autoimmunity, whereby foreign spacers guide self-targeting in a spacer-length dependent fashion. Balancing antiviral defense against autoimmunity predicts a scaling relation between spacer length and CRISPR repertoire size. We find evidence for this scaling through comparative analysis of sequenced prokaryotic genomes, and show that this association also holds at the level of CRISPR types. In contrast, the scaling is absent in strains with nonfunctional CRISPR loci. Finally, we demonstrate that stochastic spacer loss can explain variations around the scaling relation, even between strains of the same species. Our results suggest that heterologous autoimmunity is a selective factor shaping the evolution of CRISPR-Cas systems.

The role of prophages in the evolution, diversification, or virulence of the fish pathogen Flavobacterium columnare has not been studied thus far. Here, we describe a functional spontaneously inducing prophage fF4 from the F. columnare type strain ATCC 23463, which is not detectable with commonly used prophage search methods. We show that this prophage type has a global distribution and is present in strains isolated from Finland, Thailand, Japan, and North America. The virions of fF4 are myoviruses with contractile tails and infect only bacterial strains originating from Northern Finland. The fF4 resembles transposable phages by similar genome organization and several gene orthologs. Additional bioinformatic analyses reveal several species in the phylum Bacteroidetes that host a similar type of putative prophage, including bacteria that are important animal and human pathogens. Furthermore, a survey of F. columnare Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) spacers indicate a shared evolutionary history between F. columnare strains and the fF4 phage, and another putative prophage in the F. columnare strain ATCC 49512, named p49512. First, CRISPR spacer content from the two CRISPR loci (types II-C and VI-B) of the fF4 lysogen F. columnare ATCC 23463 revealed a phage terminase protein-matching spacer in the VI-B locus. This spacer is also present in two Chinese F. columnare strains. Second, CRISPR analysis revealed four F. columnare strains that contain unique spacers targeting different regions of the putative prophage p49512 in the F. columnare strain ATCC 49512, despite the geographical distance or genomovar of the different strains. This suggests a common ancestry for the F. columnare prophages and different host strains.

Background Anaerobic ammonium oxidizing bacteria (anammox bacteria) are contributing significantly to the nitrogen cycle and are successfully used in wastewater treatment. Due to the lack of complete genomes in the databases, little is known about the stability and variability of their genomes and how the genomes evolve in response to changing environments. Results Here we report the complete genome of the anammox bacterium “Candidatus Kuenenia stuttgartiensis” strain CSTR1 which was enriched planktonically in a semi-continuous stirred-tank reactor. A comparison of the genome of strain CSTR1 with the genome of “Ca. Kuenenia stuttgartiensis” MBR1 and the draft genome of KUST showed > 99% average nucleotide identity among all. Rearrangements of large genomic regions were observed, most of which were associated with transposase genes. Phylogenetic analysis suggests that strain MBR1 is more distantly related to the other two strains. Proteomic analysis of actively growing cells of strain CSTR1 (growth rate ~ 0.33 d− 1) failed to detect the annotated cytochrome cd1-type nitrite reductase (NirS) although in total 1189 proteins were found in the proteome. Yet, this NirS was expressed when strain CSTR1 was under stress or starvation (growth rate − 1). We also observed large sequence shifts in the strongly expressed S-layer protein compared to other “Ca. Kuenenia” strains, indicating the formation of hybrids of genes encoding the surface proteins. Conclusions “Ca. Kuenenia” strains appear to be relatively stable in their basic physiological traits, but show high variability in overall genome structure and surface proteins.

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.

The genus Burkholderia and its strains PAMC28687 and PAMC26561 are lichen-associated bacteria isolated from the Antarctic region. Our study is the first to provide the genome sequence of the Burkholderia sp. PAMC26561 strain. The genus Burkholderia includes bacteria that are pathogenic to plants, animals, and humans. Computational analysis of complete genomes of strains from the uncategorized Burkholderia group was performed using the NCBI databank and PATRIC (for genome sequence information) and CRISPRCasFinder (online and offline versions) software in order to predict the CRISPR loci and Cas genes. The RNAfold Webserver online software was used to predict RNA secondary structures. Our study showed that strain MSMB0852 (plasmid) possesses CRISPR-Cas system Class 2, and two lichen-associated strains, PAMC28687 (chromosome I) and PAMC26561 (chromosome I), possess CRISPR-Cas system Class 1. Additionally, only the two lichen-associated strains possess a variety of Cas genes.

Laribacter hongkongensis is a foodborne organism that is associated with gastroenteritis and diarrhea in humans. Here we describe the structural characteristics and potential function of CRISPR systems to obtain insight into the genotypic diversity of L. hongkongensis. Specifically, we analyzed the genomic content of six L. hongkongensis genomes and identified two CRISPR loci (CRISPR1 and CRISPR2) belonging to the I-F subtype of CRISPR systems. CRISPR1 was flanked on one side by cas genes and a 170 bp-long putative leader sequence, while CRISPR2 arrays located further and processed by the same cas genes. Then a combination of PCR and sequencing was used to determine the prevalence and distribution of the two CRISPR arrays in 112 L. hongkongensis strains isolated from patients, animals, and water reservoirs. In total, the CRISPR1-Cas system of complete subtype I-F was detected in 91.5% (108/118) of the isolates, whereas CRISPR2 locus existed in 72.0% (85/118). Ten strains only possessed part of the cas genes of subtype I-F and four of them with CRISPR2 array. The two loci contained highly conserved and identical direct repeat sequences which were stable in their RNA secondary structure. Additionally, 2564 total spacers including 980 unique spacers arranged in 59 alleles were identified. Homology analysis showed only 1.8% (18/980) of the spacers matched with plasmid or phage. CRISPR polymorphism present in human isolates and frog isolates was more closely related and more extensive than that of fish isolates based on spacer polymorphism. The elucidation of the structural characteristics of the CRISPR-Cas system may be helpful for further studying the specific mechanism of adaptive immunity and other biological functions mediated by CRISPR in L. hongkongensis. The conservation of CRISPR loci and hypervariable repeat-spacer arrays imply the potential for molecular typing of L. hongkongensis.

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 has 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. Co-evolution 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. A host population containing a CRISPR array variant, which did not contain 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 virus genomes revealed how selection operates at the population level. In activated sludge, it differed from the arms-race observed in nutrient rich media and resembled the fluctuating selection dynamics observed in natural environments.

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.

Background. Pseudotuberculosis is still relevant problem in medical science and public health of Russia and other countries. Typing of Y. рseudotuberculosis strains by their CRISPR systems is a perspective tool for monitoring of Yersinia populations as was shown in Y. pestis.Aims. Here we describe and compare CRISPR-Cas systems of Yersinia pseudotuberculosis strains IP32953 and IP31758 causing classic pseudotuberculosis and Far-East scarlet-like fever (FESLF) respectively.Materials and methods. Complete genomes of Y. pseudotuberculosis IP329353 and IP31758 (NC_006155 and NC_009708 respectively) were obtained from NCBI Nucleotide Database. Search; identification; and analysis of CRISPR systems were carried out by online-tools CRISPROne; CRISPRDetect; and CRISPRTarget.Results and discussion. Analyzed strains have CRISPR-Cas systems that include one set of cas-genes and arrays situated at the long distances from each other. We defined three CRISPR arrays in Y. pseudotuberculosis IP32953 by the combination of program methods. CRISPR-Cas system of this strain consist of array YP1 located near cas-genes; arrays YP2 and YP3. CRISPR-Cas system of Y. pseudotuberculosis IP31758 includes two arrays – YP1 and YP3. CRISPR systems do not share similar spacers. CRISPR systems of the analyzed strains differ in CRISPR loci and cas-protein structures that can be used as specific marks of analyzed strains.Conclusions. We suggest that acquisition of certain spacers may play a role in evolution and divergence of Y. pseudotuberculosis strains.

A CRISPR locus, defined by an array of repeat and spacer elements, constitutes a genetic record of the ceaseless battle between bacteria and viruses, showcasing the genomic integration of spacers acquired from invasive DNA. In particular, iterative spacer acquisitions represent unique evolutionary histories and are often useful for high-resolution bacterial genotyping, including comparative analysis of closely related organisms, clonal lineages, and clinical isolates. Current spacer visualization methods are typically tedious and can require manual data manipulation and curation, including spacer extraction at each CRISPR locus from genomes of interest. Here, we constructed a high-throughput extraction pipeline coupled with a local web-based visualization tool which enables CRISPR spacer and repeat extraction, rapid visualization, graphical comparison, and progressive multiple sequence alignment. We present the bioinformatic pipeline and investigate the loci of reference CRISPR-Cas systems and model organisms in 4 well-characterized subtypes. We illustrate how this analysis uncovers the evolutionary tracks and homology shared between various organisms through visual comparison of CRISPR spacers and repeats, driven through progressive alignments. Due to the ability to process unannotated genome files with minimal preparation and curation, this pipeline can be implemented promptly. Overall, this efficient high-throughput solution supports accelerated analysis of genomic data sets and enables and expedites genotyping efforts based on CRISPR loci.

Introduction:CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) loci as novel and applicable regions in prokaryotic genomes have gained great attraction in the post genomics era.Methods:These unique regions are diverse in number and sequence composition in different pathogenic bacteria and thereby can be a suitable candidate for molecular epidemiology and genotyping studies. Results:Furthermore, the arrayed structure of CRISPR loci (several unique repeats spaced with the variable sequence) and associatedcasgenes act as an active prokaryotic immune system against viral replication and conjugative elements. This property can be used as a tool for RNA editing in bioengineering studies.Conclusion:The aim of this review was to survey some details about the history, nature, and potential applications of CRISPR arrays in both genetic engineering and bacterial genotyping studies.

The adaptation phase of CRISPR–Cas immunity depends on the precise integration of short segments of foreign DNA (spacers) into a specific genomic location within the CRISPR locus by the Cas1–Cas2 integration complex. Although off-target spacer integration outside of canonical CRISPR arrays has been described in vitro, no evidence of non-specific integration activity has been found in vivo. Here, we show that non-canonical off-target integrations can occur within bacterial chromosomes at locations that resemble the native CRISPR locus by characterizing hundreds of off-target integration locations within Escherichia coli. Considering whether such promiscuous Cas1–Cas2 activity could have an evolutionary role through the genesis of neo-CRISPR loci, we combed existing CRISPR databases and available genomes for evidence of off-target integration activity. This search uncovered several putative instances of naturally occurring off-target spacer integration events within the genomes of Yersinia pestis and Sulfolobus islandicus. These results are important in understanding alternative routes to CRISPR array genesis and evolution, as well as in the use of spacer acquisition in technological applications. The examination of hundreds of off-target integrations of CRISPR spacers in Escherichia coli suggests that this process may lead to the generation of new CRISPR loci.

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&ndash, modification system were found.

Typhoid fever, caused by Salmonella enterica serovar Typhi (S. Typhi), is a global health concern and its treatment is problematic due to the rise in antimicrobial resistance (AMR). Rapid detection of patients infected with AMR positive S. Typhi is, therefore, crucial to prevent further spreading. Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated genes (CRISPR-Cas), is an adaptive immune system that initially was used for typing purposes. Later, it was discovered to play a role in defense against phages and plasmids, including ones that carry AMR genes, and, at present, it is being explored for its usage in diagnostics. Despite the availability of whole-genome sequences (WGS), very few studied the CRISPR-Cas system of S. Typhi, let alone in typing purposes or relation to AMR. In the present study, we analyzed the CRISPR-Cas system of S. Typhi using WGS data of 1059 isolates obtained from Bangladesh, India, Nepal, and Pakistan in combination with demographic data and AMR status. Our results reveal that the S. Typhi CRISPR loci can be classified into two groups: A (evidence level &gt, 2) and B (evidence level &le, 2), in which we identified a total of 47 unique spacers and 15 unique direct repeats. Further analysis of the identified spacers and repeats demonstrated specific patterns that harbored significant associations with genotype, demographic characteristics, and AMR status, thus raising the possibility of their usage as biomarkers. Potential spacer targets were identified and, interestingly, the phage-targeting spacers belonged to the group-A and plasmid-targeting spacers to the group-B CRISPR loci. Further analyses of the spacer targets led to the identification of an S. Typhi protospacer adjacent motif (PAM) sequence, TTTCA/T. New cas-genes known as DinG, DEDDh, and WYL were also discovered in the S. Typhi genome. However, a specific variant of the WYL gene was only identified in the extensively drug-resistant (XDR) lineage from Pakistan and ciprofloxacin-resistant lineage from Bangladesh. From this work, we conclude that there are strong correlations between variations identified in the S. Typhi CRISPR-Cas system and endemic AMR positive S. Typhi isolates.

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.

Spoligotyping, a graphical partial display of the CRISPR locus that can be producedin vitroorin silico, is an important tool for analyzing the diversity of givenMycobacterium tuberculosiscomplex (MTC) isolates. As other CRISPR loci, this locus is made up of an alternation between direct repeats and spacers, and flanked bycasgenes. Unveiling the genetic mechanisms of its evolution requires to have a fairly large amount of fully reconstructed loci among all MTC lineages.In this article, we point out and resolve the problem of CRISPR reconstruction based on short read sequences. We first show that more than 1/3 of the currently assembled genomes available for this complex contain a CRISPR locus erroneously reconstructed, and errors can be very significant. Second, we present a new computational method allowing this locus to be reconstructed extensively and reliablyin silicousing short read sequencing runs. Third, using this method, we describe new structural characteristics of CRISPR locus by lineages. We show how both the classical experimentalin vitroapproach and the basicin silicospoligotyping provided by existing analytic tools miss a whole diversity of this locus in MTC, by not capturing duplications, spacer and direct repeats variants, and IS6110insertion locations. This description is extended in a second article that presents general rules for the evolution of the CRISPR locus in MTC.This work opens new perspectives for a larger exploration of CRISPR loci diversity and of mechanisms involved in its evolution and its functionality.

As a naturally occurring adaptive immune system, CRISPR-Cas (clustered regularly interspersed short palindromic repeats–CRISPR-associated genes) systems are widely found in bacteria and archaea to defend against viruses. Since 2013, the application of various bacterial CRISPR-Cas systems has become very popular due to their development into targeted and programmable genome engineering tools with the ability to edit almost any genome. As the natural off-switch of CRISPR-Cas systems, anti-CRISPRs have a great potential to serve as regulators of CRISPR-Cas tools and enable safer and more controllable genome editing. This study will help understand the relative usefulness of the three bioinformatics approaches for new Acr discovery, as well as guide the future development of new bioinformatics tools to facilitate anti-CRISPR research. The thousands of Acr homologs and hundreds of new anti-CRISPR loci identified in this study will be a valuable data resource for genome engineers to search for new CRISPR-Cas regulators., Anti-CRISPR (Acr) loci/operons encode Acr proteins and Acr-associated (Aca) proteins. Forty-five Acr families have been experimentally characterized inhibiting seven subtypes of CRISPR-Cas systems. We have developed a bioinformatics pipeline to identify genomic loci containing Acr homologs and/or Aca homologs by combining three computational approaches: homology, guilt-by-association, and self-targeting spacers. Homology search found thousands of Acr homologs in bacterial and viral genomes, but most are homologous to AcrIIA7 and AcrIIA9. Investigating the gene neighborhood of these Acr homologs revealed that only a small percentage (23.0% in bacteria and 8.2% in viruses) of them have neighboring Aca homologs and thus form Acr-Aca operons. Surprisingly, although a self-targeting spacer is a strong indicator of the presence of Acr genes in a genome, a large percentage of Acr-Aca loci are found in bacterial genomes without self-targeting spacers or even without complete CRISPR-Cas systems. Additionally, for Acr homologs from genomes with self-targeting spacers, homology-based Acr family assignments do not always agree with the self-targeting CRISPR-Cas subtypes. Last, by investigating Acr genomic loci coexisting with self-targeting spacers in the same genomes, five known subtypes (I-C, I-E, I-F, II-A, and II-C) and five new subtypes (I-B, III-A, III-B, IV-A, and V-U4) of Acrs were inferred. Based on these findings, we conclude that the discovery of new anti-CRISPRs should not be restricted to genomes with self-targeting spacers and loci with Acr homologs. The evolutionary arms race of CRISPR-Cas systems and anti-CRISPR systems may have driven the adaptive and rapid gain and loss of these elements in closely related genomes. IMPORTANCE As a naturally occurring adaptive immune system, CRISPR-Cas (clustered regularly interspersed short palindromic repeats–CRISPR-associated genes) systems are widely found in bacteria and archaea to defend against viruses. Since 2013, the application of various bacterial CRISPR-Cas systems has become very popular due to their development into targeted and programmable genome engineering tools with the ability to edit almost any genome. As the natural off-switch of CRISPR-Cas systems, anti-CRISPRs have a great potential to serve as regulators of CRISPR-Cas tools and enable safer and more controllable genome editing. This study will help understand the relative usefulness of the three bioinformatics approaches for new Acr discovery, as well as guide the future development of new bioinformatics tools to facilitate anti-CRISPR research. The thousands of Acr homologs and hundreds of new anti-CRISPR loci identified in this study will be a valuable data resource for genome engineers to search for new CRISPR-Cas regulators.

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.

Type II CRISPR-Cas systems provide immunity against phages and plasmids that infect bacteria. Following infection, a short sequence of the phage genome known as the “spacer” is inserted into the CRISPR locus to capture a memory of the infection and immunize the host. Spacers are transcribed and processed into guide RNAs that direct the Cas9 nuclease to its target on the invader. Thousands of spacers are acquired to target the viral genome at multiple locations and neutralize phage mutants that evade the immunity specified by a single guide RNA. In liquid cultures, where phages and their hosts are constantly mixed, spacer diversity is generated at the population level, and a single immunization per cell is sufficient to confer robust immunity. Although rare, bacteria that acquire multiple spacers can also be found, demonstrating that type II CRISPR-Cas systems also have the capability of generating spacer diversity at the cellular level. However, conditions in which this feature is important for survival are poorly understood. Here we found that when phage infections occur on solid media, a high proportion of the surviving colonies display sectored morphologies that contain individual cells with multiple spacers. We show that this is the result of the bacteria-host co-evolution, in which the immunity provided by the initial acquired spacer is easily overcome by escaper phages that decimate all the progeny of the founder cell that do not acquire additional spacers. Our results reveal the versatility of type II CRISPR-Cas immunity, which can respond with both single or multiple spacer acquisition schemes to solve challenges presented by different environments.

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.

Objective The purpose of our study was CRISPR-based analysis of Y. pestis isolates from Georgia and neighboring countries. Introduction Particular family of tandem repeats, such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) found in a wide range of prokaryotic genomes. CRISPRs consist of highly conserved repeats interspaced with non-repetitive elements or "spacers" usually of viral origin. In the Yersinia pestis genome, three CRISPR elements YPa, YPb and YPc are found. The distribution of spacers and their arrays in Y. pestis strains is region and focus specific and can provide important information for genotyping and evolutionary research of bacteria. Methods Whole-genome sequencing (WGS) of 14 Y. pestis strains from Georgia, Armenia, Azerbaijan, Russian Federation (RF) and Kirgizstan were performed using a combination of different next generation sequencing (NGS) platforms: Illumina, PacBio and 454 technologies. Identification of spacer sequences from CRISPR loci was conducted to evaluate the genetic diversity among the isolates. The spacers arrays were acquired and analyzed. DRs and spacers were identified and extracted. The selected spacer sequences were compared against the microbial genome database in GenBank. Leader sequences were obtained for each CRISPR locus and aligned using MAFFT software. Results In all 14 sequenced genomes, three CRISPR elements were found. All alleles show the same organization, conserved 28 bp repeated sequence is interspaced with spacers, 31 or 34bp in length. A total of 18 spacers were found: seven for the YP1, six for the YP2 and five forYP3. According to the spacer arrays sequence, Biovar Microtus strains from Georgia and Armenia had the same allele sizes with the presence of seven motifs, for YP1 (a1b1c1d1e1m1n1), six motifs for YP2 (a2j2b2k2l2m2) and five for YP3 loci (a3b3c3d3e3) identical to PestoidesG (genotype 1). Spacers/spacer arrays of Biovar Medievalis strains from Georgia, Azerbaijen and Kabardino Balkaria (RF) were the same as KIM (genotype - 59). Conclusions CRISPR analysis of Georgian strains identified two independent phylogenetic groups that is in agreement with previous study data (SNP typing, IS100 fingerprinting). Biovar Microrus strains from Transcaucasian highland placed within one of the most ancient branches of the evolutionary model of Y. pestis. This study enhances our understanding and fulfills the existing data of genetic characteristics of Y. pestis strains circulating in the region.

Salmonella enterica is a major cause of bacterial food-borne disease worldwide. Immunological serotyping is the most commonly used typing method to characterize S. enterica isolates, but is time-consuming and requires expensive reagents. Here, we developed an R package CSESA (CRISPR-based Salmonella enterica Serotype Analyzer) to predict the serotype based on the CRISPR loci of S. enterica. CSESA has implemented the CRISPR typing method CLSPT and extended its coverage on diverse S. enterica serotypes. This package takes CRISPR sequences or the genome sequences as input and provides users with the predicted serotypes. CSESA has shown excellent performance with currently available sequences of S. enterica. CSESA is a convenient and useful tool for the prediction of S. enterica serotypes. The application of CSESA package can improve the efficiency of serotyping for S. enterica and reduce the burden of manpower resources. CSESA is freely available from CRAN at https://cran.r-project.org/web/packages/CSESA/ .

Over the past decade, the field of CRISPR-Cas research has received a lot of attention from the scientific community. While initially, this mostly concerned microbiologists who were fascinated by the discovery that some bacteria encode RNA-guided adaptive immune systems, this rapidly spread to other scientific disciplines following the development of groundbreaking molecular biology tools [1] and, more recently, to the public domain where the societal and ethical implications and legislation surrounding CRISPR applications are being debated. Some of the potential CRISPR applications that are currently being explored in the laboratory would involve the release of CRISPR genes into confined or open environments—for example, when CRISPR would be used to protect focal bacterial species against phage infections, when it is applied to suppress the spread of antimicrobial resistance or to control vectors of disease [2–4]. One component of the debate surrounding the societal impacts of these applications entails an assessment of the potential risks associated with these strategies (e.g. [5–7]), which requires an understanding of how CRISPR-Cas behaves in an ecological context. In this special issue, we explore this question by examining the evolutionary history of CRISPR-Cas immune systems, where they occur naturally, when they evolve and how this impacts the spread and evolution of other DNA elements. Finally, we return to the question how CRISPR-Cas may be exploited in an ecological context for the benefit of human health, and the ethical challenges that are associated with this. (a) CRISPR-Cas adaptive immune systems: a brief overview CRISPR-Cas adaptive immune systems were discovered around 15 years ago [8–11] and are estimated to exist in approximately 50% of all bacterial genomes and roughly 90% of all archaeal genomes [12]. A CRISPR immunity phenotype is genetically encoded by a so-called CRISPR locus (clustered regularly interspaced short palindromic repeats)—an array of repetitive and unique sequences (repeats and spacers, respectively), both of which are typically around 30 nt in length. Spacers are derived from (foreign) genetic elements, such as plasmids and viruses, and provide immunity to re-infection based on recognition of the cognate sequence (known as ‘protospacer’) [13–15]. Bacteria or archaea may carry a single linked array of spacers interspersed with repeats (one CRISPR locus) or multiple loci. CRISPR loci can evolve very rapidly owing to insertion of new spacers and the occasional loss of spacers or deletion of the CRISPR locus itself, which can cause very closely related strains to carry unique combinations of spacer sequences, known as a CRISPR allele. The overall length of CRISPR loci will increase and decrease with the acquisition and loss of spacers, and can vary from as little as a single spacer flanked by two repeats to hundreds of spacers and repeats [16]. As new spacers are added at the so-called leader-end of the locus, which is the sequence that contains the CRISPR promoter, CRISPR loci form an inverse chronological record of previous infections from the leader to the trailer end of the locus [17]. The extent to which different strains share the same spacer sequences in the same order (usually at the trailer end of the CRISPR locus) is commonly used to define related allele groups as a measure of their evolutionary relatedness. Apart from the genetic CRISPR memory, a functional CRISPR-Cas immune system also requires a set of CRISPR-associated genes (cas genes), which encode the protein machinery required for carrying out the immune response [18]. Cas operons vary in their cas gene composition and gene synteny, resulting in a classification of CRISPR-Cas systems into two classes, six types and 33 subtypes [19–22]. These diverse CRISPR-Cas variants differ in many of their mechanistic details, which have been discussed elsewhere [23,24], yet also have commonalities in the basic steps of the immune pathway. For example, two Cas proteins—Cas1 and Cas2—are almost invariably part of CRISPR-Cas immune systems and are responsible for inserting new spacer sequences into CRISPR arrays, sometimes assisted by other Cas proteins (reviewed in [15,25]). CRISPR transcripts are processed by either Cas proteins or housekeeping RNases [26], and the resulting processed CRISPR RNAs (crRNAs) are bound by Cas proteins to form a ribonucleoprotein complex that serves to detect and cleave complementary nucleic acid sequences [23].

Background The Proteus is one of the most common human and animal pathogens. Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR/Cas) are inheritable genetic elements found in a variety of archaea and bacteria in the evolution, providing immune function against foreign invasion. Objectives To analyze the characteristics and functions of the CRISPR/Cas system in Proteus genomes, as well as the internal and external factors affecting the system. Methods CRISPR loci were identified and divided into groups based on the repeat sequence in 96 Proteus strains by identification. Compared the RNA secondary structure and minimum free energy of CRISPR loci through bioinformatics, the evolution of cas genes, and the effects of related elements were also discussed. Results 85 CRISPR loci were identified and divided into six groups based on the sequence of repeats, and the more stable the secondary structure of RNA, the smaller the minimum free energy, the fewer base mutations in the repeat, the more stable the CRISPR and the more complete the evolution of the system. In addition, Cas1 gene can be a symbol to distinguish species to some extent. Of all the influencing factors, CRISPR/Cas had the greatest impact on plasmids. Conclusions This study examined the diversity of CRISPR/Cas system in Proteus and found statistically significant positive/negative correlations between variety factors (the RNA stability, free energy, etc.) and the CRISPR locus, which played a vital role in regulating the CRISPR/Cas system.

To study the genetic structure of the Stenotrophomonas CRISPR-Cas system using bioinformatics methods. Methods The sequence information of all Stenotrophomonas strains published in the CRISPRdb database was collected, and the CRISPR locus was analyzed using the CRISPRFinder software; All spacers were searched by BLAST platform in the PubMed database to find homologous sequences, and then the relationship between the number of spacer sequences and the number of phages was statistically analyzed. Results According to statistics, 15 confirmed CRISPR structures and 132 questionable CRISPRs were found in 26 strains of Stenotrophomonas, and the repeat sequences of CRISPR structures in different strains were more conservative. Only 1.3% of spacers were homologous with the sequences of known bacteriophages or plasmid in NCBI database. Conclusion The targeted genes of the spacer sequences are mainly from the genome of the bacteria, indicating that the evolution of the Stenotrophomonas CRISPR is related to other bacterial genes. In addition, the negative correlation between the spacer sequence and the number of phages indicates that CRISPR can prevent phage invasion. Analysis of the structure of CRISPR loci in the genome of Stenotrophomonas laid the foundation for further study of drug resistance and genomic stability.Read Complete Article at ijSciences: V82019021978 AND DOI: http://dx.doi.org/10.18483/ijSci.1978

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.

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is a bacterial defense mechanism against bacteriophages composed of two different parts: the CRISPR array and the Cas genes. The spacer acquisition is done by the adaptation module consisting of the hallmark Cas1 Cas2 proteins, which inserts new spacers into the CRISPR array. Here we aimed to describe the CRISPR-Cas system in Proteus mirabilis (P. mirabilis) isolates. CRISPR loci was observed in 30 genomic contents of 109 P. mirabilis isolates that each locus was consisted of two CRISPR arrays and each array had a different preserved leader sequences. Only the type I-E CRISPR-Cas system was common in these isolates. The source of the spacers was identified, including phages and prophages. CRISPR spacer origin analysis also identified a conserved PAM sequence of 5′-AAG-3′ nucleotide stretch. Through collecting spacers, CRISPR arrays of P. mirabilis isolates were expanded mostly by integration of bacteriophageal source of spacers. This study shows novel findings in the area of the P-mirabilis CRISPR-Cas system. In this regard, among analyzed genome of P. mirabilis isolates, Class I CRISR-Cas systems were dominant, and all belonged to type I-E. In the flanks of the CRISPR, some other elements with regulatory role were also found. A motif of 11 nt size was found to be preserved among the analyzed genome. We believe that it might has a CRISPR-Cas system transcription facilitator by targeting the Rho element.

Precise and efficient genome modifications present a great value in attempts to comprehend the roles of particular genes and other genetic elements in biological processes as well as in various pathologies. In recent years novel methods of genome modification known as genome editing, which utilize so called “programmable” nucleases, came into use. A true revolution in genome editing has been brought about by the introduction of the CRISP-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated) system, in which one of such nucleases, i.e. Cas9, plays a major role. This system is based on the elements of the bacterial and archaeal mechanism responsible for acquired immunity against phage infections and transfer of foreign genetic material. Microorganisms incorporate fragments of foreign DNA into CRISPR loci present in their genomes, which enables fast recognition and elimination of future infections. There are several types of CRISPR-Cas systems among prokaryotes but only elements of CRISPR type II are employed in genome engineering. CRISPR-Cas type II utilizes small RNA molecules (crRNA and tracrRNA) to precisely direct the effector nuclease – Cas9 – to a specific site in the genome, i.e. to the sequence complementary to crRNA. Cas9 may be used to: (i) introduce stable changes into genomes e.g. in the process of generation of knock-out and knock-in animals and cell lines, (ii) activate or silence the expression of a gene of interest, and (iii) visualize specific sites in genomes of living cells. The CRISPR-Cas-based tools have been successfully employed for generation of animal and cell models of a number of diseases, e.g. specific types of cancer. In the future, the genome editing by programmable nucleases may find wide application in medicine e.g. in the therapies of certain diseases of genetic origin and in the therapy of HIV-infected patients.

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, yielding functional and taxonomic profiles for 2.9 million spacers by aligning the spacer content to each sample’s metagenome and corresponding gene families. Spacer and repeat profiles agree qualitatively with those from isolate genomes but expand their diversity by approximately 13-fold, with the highest spacer load present in the oral microbiome. The taxonomy of spacer sequences parallels that of their source community, with functional targets enriched for viral elements. When coupled with cas gene systems, CRISPR–Cas subtypes are highly site- and taxon-specific. Our analysis provides a comprehensive collection of natural CRISPR–cas loci and targets in the human microbiome.

CRISPR (clustered regularly interspaced short palindromic repeats) loci and their associated (cas) genes encode an adaptive immune system that protects prokaryotes from viral1 and plasmid2 invaders. Following viral (phage) infection, a small fraction of the prokaryotic cells are able to integrate a small sequence of the invader’s genome into the CRISPR array1. These sequences, known as spacers, are transcribed and processed into small CRISPR RNA guides3–5 that associate with Cas nucleases to specify a viral target for destruction6–9. Although CRISPR−cas loci are widely distributed throughout microbial genomes and often display hallmarks of horizontal gene transfer10–12, the drivers of CRISPR dissemination remain unclear. Here, we show that spacers can recombine with phage target sequences to mediate a form of specialized transduction of CRISPR elements. Phage targets in phage 85, ΦNM1, ΦNM4 and Φ12 can recombine with spacers in either chromosomal or plasmid-borne CRISPR loci in Staphylococcus, leading to either the transfer of CRISPR-adjacent genes or the propagation of acquired immunity to other bacteria in the population, respectively. Our data demonstrate that spacer sequences not only specify the targets of Cas nucleases but also can promote horizontal gene transfer. CRISPR spacers can recombine with phage target sequences to mediate a form of specialized transduction that can promote transfer of CRISPR elements to other bacteria in the population.

Lactobacillus helveticus is a homofermentative thermophilic lactic acid bacteria that is mainly used in the manufacture of Swiss type and long-ripened Italian hard cheeses. In this study, the presence of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) were analysed in 25 L. helveticus genomes and identified in 23 of these genomes. A total of 40 CRISPR loci were identified and classified into five main families based on CRISPR repeats: Ldbu1, Lsal1, Lhel1, Lhel2 and a new repeat family named Lhel3. Spacers had a size between 30 and 40 bp whereas repeats have an average size of 30 bp, with three longer repeats. The analysis displayed the presence of conserved spacers in 23 of the 40 CRISPR loci. A geographical distribution of L. helveticus isolates with similar CRISPR spacer array profiles were not observed. Based on the presence of the signature protein Cas3, all CRISPR loci belonged to Type I. This analysis demonstrated a great CRISPR array variability within L. helveticus, which could be a useful tool for genotypic strain differentiation. A next step will be to understand the possible role of CRISPR/Cas system for the resistance of L. helveticus to phage infection. SIGNIFICANCE AND IMPACT OF THE STUDY: Lactobacillus helveticus, a lactic acid bacteria species widely used as starter culture in the dairy industry has recently also gained importance as health-promoting culture in probiotic and nutraceutical food products. The CRISPR/Cas system, a well-known molecular mechanism that provides adaptive immunity against exogenous genetic elements such as bacteriophages and plasmids in bacteria, was recently found in this species. In this study, we investigated the presence and genetic heterogeneity of CRISPR loci in 25 L. helveticus genomes. The results presented here represent an important step on the way to manage phage resistance, plasmid uptake and genome editing in this species.

The interspaced short palindromic repeats (CRISPR) system is an immune system widely distributed in prokaryotes, resisting the invasion of the foreign mobile genetic elements like phages or plasmids. In this study, we present the comparative analysis of 182 CRISPR loci found in 46 publicly available complete genome sequences of Streptomyces. Overall, nine direct repeats (DRs) groups are identified while all the 2104 spacers are divided into three main groups according to the multiple sequence alignment. Only 11 spacers are identical with parts of 10 plasmid sequences, which indicates a possible origin. The cas gene clusters near the CRISPR arrays are found to mainly belong to the I-E subtype. These CRISPR loci might play an important role in the genome evolution of Streptomyces.

Genome rearrangements have played an important role in the evolution of Yersinia pestis from its progenitor Yersinia pseudotuberculosis. Traditional phylogenetic trees for Y. pestis based on sequence comparison have short internal branches and low bootstrap supports as only a small number of nucleotide substitutions have occurred. On the other hand, even a small number of genome rearrangements may resolve topological ambiguities in a phylogenetic tree. We reconstructed the evolutionary history of genome rearrangements in Y. pestis. We also reconciled phylogenetic trees for each of the three CRISPR-loci to obtain an integrated scenario of the CRISPR-cassette evolution. We detected numerous parallel inversions and gain/loss events by the analysis of contradictions between the obtained evolutionary trees. We also tested the hypotheses that large within-replichore inversions tend to be balanced by subsequent reversal events and that the core genes less frequently switch the chain by inversions. Both predictions were not confirmed. Our data indicate that an integrated analysis of sequence-based and inversion-based trees enhances the resolution of phylogenetic reconstruction. In contrast, reconstructions of strain relationships based on solely CRISPR loci may not be reliable, as the history is obscured by large deletions, obliterating the order of spacer gains. Similarly, numerous parallel gene losses preclude reconstruction of phylogeny based on gene content.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) system is a novel type of innate defense system in prokaryotes for destruction of exogenous elements. To gain further insight into behavior and organization of the system, the extensive analysis of the available sequenced genomes is necessary. The dynamic nature of CRISPR loci is possibly valuable for typing and relative analyses of strains and microbial population. There are a few orderly bioinformatics investigations about the structure of CRISPR sequences in the Escherichia coli strains. In this study, 57 CRISPR loci were selected from 32 Escherichia coli strains to investigate their structural characteristics and potential functions using bioinformatics tools. Our results showed that most strains contained several loci that mainly included conserved direct repeats, while the spacers were highly variable. Moreover, RNA analysis of the sequences indicated that all loci could form stable RNA secondary structures and showed homology mostly with phages compared to plasmids. Only three strains included cas genes around their loci.

Limited by culture-dependent methods the number of viruses identified from thermophilic Archaea and Bacteria is still very small. In this study we retrieved viral sequences from six hot spring metagenomes isolated worldwide, revealing a wide distribution of four archaeal viral families, Ampullaviridae, Bicaudaviridae, Lipothrixviridae and Rudiviridae. Importantly, we identified 10 complete or near complete viral genomes allowing, for the first time, an assessment of genome conservation and evolution of the Ampullaviridae family as well as Sulfolobus Monocaudavirus 1 (SMV1)-related viruses. Among the novel genomes, one belongs to a putative thermophilic virus infecting the bacterium Hydrogenobaculum, for which no virus has been reported in the literature. Moreover, a high viral diversity was observed in the metagenomes, especially among the Lipothrixviridae, as indicated by the large number of unique contigs and the lack of a completely assembled genome for this family. This is further supported by the large number of novel genes in the complete and partial genomes showing no sequence similarities to public databases. CRISPR analysis revealed hundreds of novel CRISPR loci and thousands of novel CRISPR spacers from each metagenome, reinforcing the notion of high viral diversity in the thermal environment.

International audience; The natural history of tuberculosis may be tackled by various means, among which the record of molecular scars that have been registered by the Mycobacterium tuberculosis complex (MTBC) genomes transmitted from patient to patient for tens of thousands years and possibly more. Recently discovered polymorphic loci, the CRISPR sequences, are indirect witnesses of the historical phage-bacteria struggle, and may be related to the time when the ancestor of today's tubercle bacilli were environmental bacteria, i.e. before becoming intracellular parasites. In this article, we present what are CRISPRs and try to summarize almost 20 years of research results obtained using the genetic diversity of the CRISPR loci in MTBC as a perspective for studying new models. We show that the study of the diversity of CRISPR sequences, thanks to «spoligotyping», has played a great role in our global understanding of the population structure of MTBC.

The adaptation phase of CRISPR-Cas immunity depends on the precise integration of short segments of foreign DNA (spacers) into a specific genomic location within the CRISPR locus by the Cas1-Cas2 integration complex. Although off-target spacer integration outside of canonical CRISPR arrays has been described in vitro, no evidence of non-specific integration activity has been found in vivo. Here, we show that non-canonical off-target integrations can occur within bacterial chromosomes at locations that resemble the native CRISPR locus by characterizing hundreds of off-target integration locations within Escherichia coli. Considering whether such promiscuous Cas1-Cas2 activity could have an evolutionary role through the genesis of neo-CRISPR loci, we combed existing CRISPR databases and available genomes for evidence of off-target integration activity. This search uncovered several putative instances of naturally occurring off-target spacer integration events within the genomes of Yersinia pestis and Sulfolobus islandicus. These results are important in understanding alternative routes to CRISPR array genesis and evolution, as well as in the use of spacer acquisition in technological applications.

Diverse CRISPR-Cas systems provide adaptive immunity in many bacteria and most archaea, via a DNA-encoded, RNA-mediated, nucleic-acid targeting mechanism. Over time, CRISPR loci expand via iterative uptake of invasive DNA sequences into the CRISPR array during the adaptation process. These genetic vaccination cards thus provide insights into the exposure of strains to phages and plasmids in space and time, revealing the historical predatory exposure of a strain. These genetic loci thus constitute a unique basis for genotyping of strains, with potential of resolution at the strain-level. Here, we investigate the occurrence and diversity of CRISPR-Cas systems in the genomes of various Bifidobacterium longum strains across three sub-species. Specifically, we analyzed the genomic content of 66 genomes belonging to B. longum subsp. longum, B. longum subsp. infantis and B. longum subsp. suis, and identified 25 strains that carry 29 total CRISPR-Cas systems. We identify various Type I and Type II CRISPR-Cas systems that are widespread in this species, notably I-C, I-E, and II-C. Noteworthy, Type I-C systems showed extended CRISPR arrays, with extensive spacer diversity. We show how these hypervariable loci can be used to gain insights into strain origin, evolution and phylogeny, and can provide discriminatory sequences to distinguish even clonal isolates. By investigating CRISPR spacer sequences, we reveal their origin and implicate phages and prophages as drivers of CRISPR immunity expansion in this species, with redundant targeting of select prophages. Analysis of CRISPR spacer origin also revealed novel PAM sequences. Our results suggest that CRISPR-Cas immune systems are instrumental in mounting diversified viral resistance in B. longum, and show that these sequences are useful for typing across three subspecies, This study was funded by start-up funds from North Carolina State University and the North Carolina Ag Foundation

Background Much effort is underway to build and upgrade databases and tools related to occurrence, diversity, and characterization of CRISPR-Cas systems. As microbial communities and their genome complements are unearthed, much emphasis has been placed on details of individual strains and model systems within the CRISPR-Cas classification, and that collection of information as a whole affords the opportunity to analyze CRISPR-Cas systems from a quantitative perspective to gain insight into distribution of CRISPR array sizes across the different classes, types and subtypes. CRISPR diversity, nomenclature, occurrence, and biological functions have generated a plethora of data that created a need to understand the size and distribution of these various systems to appreciate their features and complexity. Results By utilizing a statistical framework and visual analytic techniques, we have been able to test several hypotheses about CRISPR loci in bacterial class I systems. Quantitatively, though CRISPR loci can expand to hundreds of spacers, the mean and median sizes are 40 and 25, respectively, reflecting rather modest acquisition and/or retention overall. Histograms uncovered that CRISPR array size displayed a parametric distribution, which was confirmed by a goodness-of fit test. Mapping the frequency of CRISPR loci on a standardized chromosome plot revealed that CRISPRs have a higher probability of occurring at clustered locations along the positive or negative strand. Lastly, when multiple arrays occur in a particular system, the size of a particular CRISPR array varies with its distance from the cas operon, reflecting acquisition and expansion biases. Conclusions This study establishes that bacterial Class I CRISPR array size tends to follow a geometric distribution; these CRISPRs are not randomly distributed along the chromosome; and the CRISPR array closest to the cas genes is typically larger than loci in trans. Overall, we provide an analytical framework to understand the features and behavior of CRISPR-Cas systems through a quantitative lens. Reviewers This article was reviewed by Eugene Koonin (NIH-NCBI) and Uri Gophna (Tel Aviv University). Electronic supplementary material The online version of this article (doi:10.1186/s13062-017-0193-2) contains supplementary material, which is available to authorized users.

Thermophilic environments represent an interesting niche. Among thermophiles, the genus Thermus is among the most studied genera. In this study, we have sequenced the genome of Thermus parvatiensis strain RL, a thermophile isolated from Himalayan hot water springs (temperature >96°C) using PacBio RSII SMRT technique. The small genome (2.01 Mbp) comprises a chromosome (1.87 Mbp) and a plasmid (143 Kbp), designated in this study as pTP143. Annotation revealed a high number of repair genes, a squeezed genome but containing highly plastic plasmid with transposases, integrases, mobile elements and hypothetical proteins (44%). We performed a comparative genomic study of the group Thermus with an aim of analysing the phylogenetic relatedness as well as niche specific attributes prevalent among the group. We compared the reference genome RL with 16 Thermus genomes to assess their phylogenetic relationships based on 16S rRNA gene sequences, average nucleotide identity (ANI), conserved marker genes (31 and 400), pan genome and tetranucleotide frequency. The core genome of the analyzed genomes contained 1,177 core genes and many singleton genes were detected in individual genomes, reflecting a conserved core but adaptive pan repertoire. We demonstrated the presence of metagenomic islands (chromosome:5, plasmid:5) by recruiting raw metagenomic data (from the same niche) against the genomic replicons of T. parvatiensis. We also dissected the CRISPR loci wide all genomes and found widespread presence of this system across Thermus genomes. Additionally, we performed a comparative analysis of competence loci wide Thermus genomes and found evidence for recent horizontal acquisition of the locus and continued dispersal among members reflecting that natural competence is a beneficial survival trait among Thermus members and its acquisition depicts unending evolution in order to accomplish optimal fitness.

Antagonistic coevolution of parasite infectivity and host resistance may alter the biological functionality of species, yet these dynamics in nature are still poorly understood. Here we show the molecular details of a long-term phage–bacterium arms race in the environment. Bacteria (Flavobacterium columnare) are generally resistant to phages from the past and susceptible to phages isolated in years after bacterial isolation. Bacterial resistance selects for increased phage infectivity and host range, which is also associated with expansion of phage genome size. We identified two CRISPR loci in the bacterial host: a type II-C locus and a type VI-B locus. While maintaining a core set of conserved spacers, phage-matching spacers appear in the variable ends of both loci over time. The spacers mostly target the terminal end of the phage genomes, which also exhibit the most variation across time, resulting in arms-race-like changes in the protospacers of the coevolving phage population., Arms races between phage and bacteria are well known from lab experiments, but insight from field systems is limited. Here, the authors show changes in the resistance and CRISPR loci of bacteria and the infectivity, host range and genome size of phage over multiple years in an aquaculture environment.

Listeria monocytogenes, an important food-borne pathogen, causes high mortality rate of listeriosis. Pan-genomic comparisons revealed the species genome of L. monocytogenes is highly stable but not completely clonal. The population structure of this species displays at least four evolutionary lineages (I-IV). Isolates of different lineages displayed distinct genetic, phenotypic and ecologic characteristics, which appear to affect their ability to be transmitted through foods and to cause human disease, as well as their ability to thrive in markedly phage-rich environments. CRISPR (clustered regularly interspaced short palindrome repeats), a recently described adaptive immunity system, not only confers defense against invading elements derived from bacteriophages or plasmids in many bacteria and archaeal, but also displays strains-level variations in almost any given endowed species. This work was aimed to investigate CRISPR diversity in L. monocytogenes strains of different lineages and estimated the potential practicability of the CRISPR-based approach to resolve this species' biodiversity. Only a third of strains contained all three CRISPR loci (here defined as LMa, LMb and LMc) at same time. Combined the strain-level variations in presence/absence of each CRISPR locus and its relative size and spacer arrangements, a total of 29 CRISPR genotypes and 11 groups were defined within a collection of 128 strains covering all serotypes. The CRISPR-based approach showed powerful ability to subtype the more commonly food-borne isolates of serotype 1/2a (lineage II) and serotypes 1/2b (lineage I), but limited by the absence of typical CRISPR structure in many lineage I isolates. Strikingly, we found a long associated cas1 gene as well as two self-targeting LMb spacers accidently homologous with endogenous genes in a fraction of serotype 1/2a isolations, demonstrated that CRISPR I B system might involve in bacterial physiology besides antiviral immunity.

The CRISPR (clusters of regularly interspaced short palindromic repeats)–Cas adaptive immune system is an important defense system in bacteria, providing targeted defense against invasions of foreign nucleic acids. CRISPR–Cas systems consist of CRISPR loci and cas (CRISPR-associated) genes: sequence segments of invaders are incorporated into host genomes at CRISPR loci to generate specificity, while adjacent cas genes encode proteins that mediate the defense process. We pursued an integrated approach to identifying putative cas genes from genomes and metagenomes, combining similarity searches with genomic neighborhood analysis. Application of our approach to bacterial genomes and human microbiome datasets allowed us to significantly expand the collection of cas genes: the sequence space of the Cas9 family, the key player in the recently engineered RNA-guided platforms for genome editing in eukaryotes, is expanded by at least two-fold with metagenomic datasets. We found genes in cas loci encoding other functions, for example, toxins and antitoxins, confirming the recently discovered potential of coupling between adaptive immunity and the dormancy/suicide systems. We further identified 24 novel Cas families; one novel family contains 20 proteins, all identified from the human microbiome datasets, illustrating the importance of metagenomics projects in expanding the diversity of cas genes.