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Horvath, Philippe, Dennis A. Romero, Anne-Claire Coûté-Monvoisin, Melissa Richards, Hélène Deveau, Sylvain Moineau, Patrick Boyaval, Christophe Fremaux und Rodolphe Barrangou. „Diversity, Activity, and Evolution of CRISPR Loci in Streptococcus thermophilus“. Journal of Bacteriology 190, Nr. 4 (07.12.2007): 1401–12. http://dx.doi.org/10.1128/jb.01415-07.
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ABSTRACT Clustered regularly interspaced short palindromic repeats (CRISPR) are hypervariable loci widely distributed in prokaryotes that provide acquired immunity against foreign genetic elements. Here, we characterize a novel Streptococcus thermophilus locus, CRISPR3, and experimentally demonstrate its ability to integrate novel spacers in response to bacteriophage. Also, we analyze CRISPR diversity and activity across three distinct CRISPR loci in several S. thermophilus strains. We show that both CRISPR repeats and cas genes are locus specific and functionally coupled. A total of 124 strains were studied, and 109 unique spacer arrangements were observed across the three CRISPR loci. Overall, 3,626 spacers were analyzed, including 2,829 for CRISPR1 (782 unique), 173 for CRISPR2 (16 unique), and 624 for CRISPR3 (154 unique). Sequence analysis of the spacers revealed homology and identity to phage sequences (77%), plasmid sequences (16%), and S. thermophilus chromosomal sequences (7%). Polymorphisms were observed for the CRISPR repeats, CRISPR spacers, cas genes, CRISPR motif, locus architecture, and specific sequence content. Interestingly, CRISPR loci evolved both via polarized addition of novel spacers after exposure to foreign genetic elements and via internal deletion of spacers. We hypothesize that the level of diversity is correlated with relative CRISPR activity and propose that the activity is highest for CRISPR1, followed by CRISPR3, while CRISPR2 may be degenerate. Globally, the dynamic nature of CRISPR loci might prove valuable for typing and comparative analyses of strains and microbial populations. Also, CRISPRs provide critical insights into the relationships between prokaryotes and their environments, notably the coevolution of host and viral genomes.
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Toro, Magaly, Guojie Cao, Wenting Ju, Marc Allard, Rodolphe Barrangou, Shaohua Zhao, Eric Brown und Jianghong Meng. „Association of Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) Elements with Specific Serotypes and Virulence Potential of Shiga Toxin-Producing Escherichia coli“. Applied and Environmental Microbiology 80, Nr. 4 (13.12.2013): 1411–20. http://dx.doi.org/10.1128/aem.03018-13.
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ABSTRACTShiga toxin-producingEscherichia coli(STEC) strains (n= 194) representing 43 serotypes andE. coliK-12 were examined for clustered regularly interspaced short palindromic repeat (CRISPR) arrays to study genetic relatedness among STEC serotypes. A subset of the strains (n= 81) was further analyzed for subtype I-Ecasand virulence genes to determine a possible association of CRISPR elements with potential virulence. Four types of CRISPR arrays were identified. CRISPR1 and CRISPR2 were present in all strains tested; 1 strain also had both CRISPR3 and CRISPR4, whereas 193 strains displayed a short, combined array, CRISPR3-4. A total of 3,353 spacers were identified, representing 528 distinct spacers. The average length of a spacer was 32 bp. Approximately one-half of the spacers (54%) were unique and found mostly in strains of less common serotypes. Overall, CRISPR spacer contents correlated well with STEC serotypes, and identical arrays were shared between strains with the same H type (O26:H11, O103:H11, and O111:H11). There was no association identified between the presence of subtype I-Ecasand virulence genes, but the total number of spacers had a negative correlation with potential pathogenicity (P< 0.05). Fewer spacers were found in strains that had a greater probability of causing outbreaks and disease than in those with lower virulence potential (P< 0.05). The relationship between the CRISPR-cassystem and potential virulence needs to be determined on a broader scale, and the biological link will need to be established.
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Achigar, Rodrigo, Martina Scarrone, Geneviève M. Rousseau, Cécile Philippe, Felipe Machado, Valentina Duvós, María Pía Campot et al. „Ectopic Spacer Acquisition in Streptococcus thermophilus CRISPR3 Array“. Microorganisms 9, Nr. 3 (01.03.2021): 512. http://dx.doi.org/10.3390/microorganisms9030512.
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Streptococcus thermophilus relies heavily on two type II-A CRISPR-Cas systems, CRISPR1 and CRISPR3, to resist siphophage infections. One hallmark of these systems is the integration of a new spacer at the 5′ end of the CRISPR arrays following phage infection. However, we have previously shown that ectopic acquisition of spacers can occur within the CRISPR1 array. Here, we present evidence of the acquisition of new spacers within the array of CRISPR3 of S. thermophilus. The analysis of randomly selected bacteriophage-insensitive mutants of the strain Uy01 obtained after phage infection, as well as the comparison with other S. thermophilus strains with similar CRISPR3 content, showed that a specific spacer within the array could be responsible for misguiding the adaptation complex. These results also indicate that while the vast majority of new spacers are added at the 5′ end of the CRISPR array, ectopic spacer acquisition is a common feature of both CRISPR1 and CRISPR3 systems in S. thermophilus, and it can still provide phage resistance. Ectopic spacer acquisition also appears to have occurred naturally in some strains of Streptococcus pyogenes, suggesting that it is a general phenomenon, at least in type II-A systems.
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van der Ploeg, Jan R. „Analysis of CRISPR in Streptococcus mutans suggests frequent occurrence of acquired immunity against infection by M102-like bacteriophages“. Microbiology 155, Nr. 6 (01.06.2009): 1966–76. http://dx.doi.org/10.1099/mic.0.027508-0.
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Clustered regularly interspaced short palindromic repeats (CRISPR) consist of highly conserved direct repeats interspersed with variable spacer sequences. They can protect bacteria against invasion by foreign DNA elements. The genome sequence of Streptococcus mutans strain UA159 contains two CRISPR loci, designated CRISPR1 and CRISPR2. The aims of this study were to analyse the organization of CRISPR in further S. mutans strains and to investigate the importance of CRISPR in acquired immunity to M102-like phages. The sequences of CRISPR1 and CRISPR2 arrays were determined for 29 S. mutans strains from different persons. More than half of the CRISPR1 spacers and about 35 % of the CRISPR2 spacers showed sequence similarity with the genome sequence of M102, a virulent siphophage specific for S. mutans. Although only a few spacers matched the phage sequence completely, most of the mismatches had no effect on the amino acid sequences of the phage-encoded proteins. The results suggest that S. mutans is often attacked by M102-like bacteriophages, and that its acquisition of novel phage-derived CRISPR sequences goes along with the presence of S. mutans phages in the environment. Analysis of CRISPR1 of M102-resistant mutants of S. mutans OMZ 381 showed that some of them had acquired novel spacers, and the sequences of all but one of these matched the phage M102 genome sequence. This suggests that the acquisition of the spacers contributed to the resistance against phage infection. However, since not all resistant mutants had new spacers, and since the removal of the CRISPR1 array in one of the mutants and in wild-type strains did not lead to loss of resistance to infection by M102, the acquisition of resistance must be based on further elements as well.
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Serbanescu, M. A., M. Cordova, K. Krastel, R. Flick, N. Beloglazova, A. Latos, A. F. Yakunin, D. B. Senadheera und D. G. Cvitkovitch. „Role of the Streptococcus mutans CRISPR-Cas Systems in Immunity and Cell Physiology“. Journal of Bacteriology 197, Nr. 4 (08.12.2014): 749–61. http://dx.doi.org/10.1128/jb.02333-14.
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CRISPR-Cas systems provide adaptive microbial immunity against invading viruses and plasmids. The cariogenic bacteriumStreptococcus mutansUA159 has two CRISPR-Cas systems: CRISPR1 (type II-A) and CRISPR2 (type I-C) with several spacers from both CRISPR cassettes matching sequences of phage M102 or genomic sequences of otherS. mutans. The deletion of thecasgenes of CRISPR1 (ΔC1S), CRISPR2 (ΔC2E), or both CRISPR1+2 (ΔC1SC2E) or the removal of spacers 2 and 3 (ΔCR1SP13E) inS. mutansUA159 did not affect phage sensitivity when challenged with virulent phage M102. Using plasmid transformation experiments, we demonstrated that the CRISPR1-Cas system inhibits transformation ofS. mutansby the plasmids matching the spacers 2 and 3. Functional analysis of thecasdeletion mutants revealed that in addition to a role in plasmid targeting, both CRISPR systems also contribute to the regulation of bacterial physiology inS. mutans. Compared to wild-type cells, the ΔC1S strain displayed diminished growth under cell membrane and oxidative stress, enhanced growth under low pH, and had reduced survival under heat shock and DNA-damaging conditions, whereas the ΔC2E strain exhibited increased sensitivity to heat shock. Transcriptional analysis revealed that the two-component signal transduction system VicR/K differentially modulates expression ofcasgenes within CRISPR-Cas systems, suggesting that VicR/K might coordinate the expression of two CRISPR-Cas systems. Collectively, we providein vivoevidence that the type II-A CRISPR-Cas system ofS. mutansmay be targeted to manipulate its stress response and to influence the host to control the uptake and dissemination of antibiotic resistance genes.
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Pavlova, Yekaterina S., David Paez-Espino, Andrew Yu Morozov und Ilya S. Belalov. „Searching for fat tails in CRISPR-Cas systems: Data analysis and mathematical modeling“. PLOS Computational Biology 17, Nr. 3 (26.03.2021): e1008841. http://dx.doi.org/10.1371/journal.pcbi.1008841.
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Understanding CRISPR-Cas systems—the adaptive defence mechanism that about half of bacterial species and most of archaea use to neutralise viral attacks—is important for explaining the biodiversity observed in the microbial world as well as for editing animal and plant genomes effectively. The CRISPR-Cas system learns from previous viral infections and integrates small pieces from phage genomes called spacers into the microbial genome. The resulting library of spacers collected in CRISPR arrays is then compared with the DNA of potential invaders. One of the most intriguing and least well understood questions about CRISPR-Cas systems is the distribution of spacers across the microbial population. Here, using empirical data, we show that the global distribution of spacer numbers in CRISPR arrays across multiple biomes worldwide typically exhibits scale-invariant power law behaviour, and the standard deviation is greater than the sample mean. We develop a mathematical model of spacer loss and acquisition dynamics which fits observed data from almost four thousand metagenomes well. In analogy to the classical ‘rich-get-richer’ mechanism of power law emergence, the rate of spacer acquisition is proportional to the CRISPR array size, which allows a small proportion of CRISPRs within the population to possess a significant number of spacers. Our study provides an alternative explanation for the rarity of all-resistant super microbes in nature and why proliferation of phages can be highly successful despite the effectiveness of CRISPR-Cas systems.
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Deveau, Hélène, Rodolphe Barrangou, Josiane E. Garneau, Jessica Labonté, Christophe Fremaux, Patrick Boyaval, Dennis A. Romero, Philippe Horvath und Sylvain Moineau. „Phage Response to CRISPR-Encoded Resistance in Streptococcus thermophilus“. Journal of Bacteriology 190, Nr. 4 (07.12.2007): 1390–400. http://dx.doi.org/10.1128/jb.01412-07.
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ABSTRACT Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated genes are linked to a mechanism of acquired resistance against bacteriophages. Bacteria can integrate short stretches of phage-derived sequences (spacers) within CRISPR loci to become phage resistant. In this study, we further characterized the efficiency of CRISPR1 as a phage resistance mechanism in Streptococcus thermophilus. First, we show that CRISPR1 is distinct from previously known phage defense systems and is effective against the two main groups of S. thermophilus phages. Analyses of 30 bacteriophage-insensitive mutants of S. thermophilus indicate that the addition of one new spacer in CRISPR1 is the most frequent outcome of a phage challenge and that the iterative addition of spacers increases the overall phage resistance of the host. The added new spacers have a size of between 29 to 31 nucleotides, with 30 being by far the most frequent. Comparative analysis of 39 newly acquired spacers with the complete genomic sequences of the wild-type phages 2972, 858, and DT1 demonstrated that the newly added spacer must be identical to a region (named proto-spacer) in the phage genome to confer a phage resistance phenotype. Moreover, we found a CRISPR1-specific sequence (NNAGAAW) located downstream of the proto-spacer region that is important for the phage resistance phenotype. Finally, we show through the analyses of 20 mutant phages that virulent phages are rapidly evolving through single nucleotide mutations as well as deletions, in response to CRISPR1.
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Bolotin, Alexander, Benoit Quinquis, Alexei Sorokin und S. Dusko Ehrlich. „Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin“. Microbiology 151, Nr. 8 (01.08.2005): 2551–61. http://dx.doi.org/10.1099/mic.0.28048-0.
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Numerous prokaryote genomes contain structures known as clustered regularly interspaced short palindromic repeats (CRISPRs), composed of 25–50 bp repeats separated by unique sequence spacers of similar length. CRISPR structures are found in the vicinity of four genes named cas1 to cas4. In silico analysis revealed another cluster of three genes associated with CRISPR structures in many bacterial species, named here as cas1B, cas5 and cas6, and also revealed a certain number of spacers that have homology with extant genes, most frequently derived from phages, but also derived from other extrachromosomal elements. Sequence analysis of CRISPR structures from 24 strains of Streptococcus thermophilus and Streptococcus vestibularis confirmed the homology of spacers with extrachromosomal elements. Phage sensitivity of S. thermophilus strains appears to be correlated with the number of spacers in the CRISPR locus the strain carries. The authors suggest that the spacer elements are the traces of past invasions by extrachromosomal elements, and hypothesize that they provide the cell immunity against phage infection, and more generally foreign DNA expression, by coding an anti-sense RNA. The presence of gene fragments in CRISPR structures and the nuclease motifs in cas genes of both cluster types suggests that CRISPR formation involves a DNA degradation step.
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Heussler, Gary E., Jon L. Miller, Courtney E. Price, Alan J. Collins und George A. O'Toole. „Requirements for Pseudomonas aeruginosa Type I-F CRISPR-Cas Adaptation Determined Using a Biofilm Enrichment Assay“. Journal of Bacteriology 198, Nr. 22 (29.08.2016): 3080–90. http://dx.doi.org/10.1128/jb.00458-16.
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ABSTRACTCRISPR (clustered regularly interspaced short palindromic repeat)-Cas (CRISPR-associated protein) systems are diverse and found in many archaea and bacteria. These systems have mainly been characterized as adaptive immune systems able to protect against invading mobile genetic elements, including viruses. The first step in this protection is acquisition of spacer sequences from the invader DNA and incorporation of those sequences into the CRISPR array, termed CRISPR adaptation. Progress in understanding the mechanisms and requirements of CRISPR adaptation has largely been accomplished using overexpression ofcasgenes or plasmid loss assays; little work has focused on endogenous CRISPR-acquired immunity from viral predation. Here, we developed a new biofilm-based assay system to enrich forPseudomonas aeruginosastrains with new spacer acquisition. We used this assay to demonstrate thatP. aeruginosarapidly acquires spacers protective against DMS3vir, an engineered lytic variant of the Mu-like bacteriophage DMS3, through primed CRISPR adaptation from spacers present in the native CRISPR2 array. We found that for theP. aeruginosatype I-F system, thecas1gene is required for CRISPR adaptation,recGcontributes to (but is not required for) primed CRISPR adaptation,recDis dispensable for primed CRISPR adaptation, and finally, the ability of a putative priming spacer to prime can vary considerably depending on the specific sequences of the spacer.IMPORTANCEOur understanding of CRISPR adaptation has expanded largely through experiments in type I CRISPR systems using plasmid loss assays, mutants ofEscherichia coli, orcas1-cas2overexpression systems, but there has been little focus on studying the adaptation of endogenous systems protecting against a lytic bacteriophage. Here we describe a biofilm system that allowsP. aeruginosato rapidly gain spacers protective against a lytic bacteriophage. This approach has allowed us to probe the requirements for CRISPR adaptation in the endogenous type I-F system ofP. aeruginosa. Our data suggest that CRISPR-acquired immunity in a biofilm may be one reason that manyP. aeruginosastrains maintain a CRISPR-Cas system.
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Lopatina, Anna, Sofia Medvedeva, Daria Artamonova, Matvey Kolesnik, Vasily Sitnik, Yaroslav Ispolatov und Konstantin Severinov. „Natural diversity of CRISPR spacers of Thermus : evidence of local spacer acquisition and global spacer exchange“. Philosophical Transactions of the Royal Society B: Biological Sciences 374, Nr. 1772 (25.03.2019): 20180092. http://dx.doi.org/10.1098/rstb.2018.0092.
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We investigated the diversity of CRISPR spacers of Thermus communities from two locations in Italy, two in Chile and one location in Russia. Among the five sampling sites, a total of more than 7200 unique spacers belonging to different CRISPR-Cas systems types and subtypes were identified. Most of these spacers are not found in CRISPR arrays of sequenced Thermus strains. Comparison of spacer sets revealed that samples within the same area (separated by few to hundreds of metres) have similar spacer sets, which appear to be largely stable at least over the course of several years. While at further distances (hundreds of kilometres and more) the similarity of spacer sets is decreased, there are still multiple common spacers in Thermus communities from different continents. The common spacers can be reconstructed in identical or similar CRISPR arrays, excluding their independent appearance and suggesting an extensive migration of thermophilic bacteria over long distances. Several new Thermus phages were isolated in the sampling sites. Mapping of spacers to bacteriophage sequences revealed examples of local acquisition of spacers from some phages and distinct patterns of targeting of phage genomes by different CRISPR-Cas systems. This article is part of a discussion meeting issue ‘The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems’.
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Mojica, F. J. M., C. Díez-Villaseñor, J. García-Martínez und C. Almendros. „Short motif sequences determine the targets of the prokaryotic CRISPR defence system“. Microbiology 155, Nr. 3 (01.03.2009): 733–40. http://dx.doi.org/10.1099/mic.0.023960-0.
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Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated CRISPR-associated sequence (CAS) proteins constitute a novel antiviral defence system that is widespread in prokaryotes. Repeats are separated by spacers, some of them homologous to sequences in mobile genetic elements. Although the whole process involved remains uncharacterized, it is known that new spacers are incorporated into CRISPR loci of the host during a phage challenge, conferring specific resistance against the virus. Moreover, it has been demonstrated that such interference is based on small RNAs carrying a spacer. These RNAs would guide the defence apparatus to foreign molecules carrying sequences that match the spacers. Despite this essential role, the spacer uptake mechanism has not been addressed. A first step forward came from the detection of motifs associated with spacer precursors (proto-spacers) of Streptococcus thermophilus, revealing a specific recognition of donor sequences in this species. Here we show that the conservation of proto-spacer adjacent motifs (PAMs) is a common theme for the most diverse CRISPR systems. The PAM sequence depends on the CRISPR-CAS variant, implying that there is a CRISPR-type-specific (motif-directed) choice of the spacers, which subsequently determines the interference target. PAMs also direct the orientation of spacers in the repeat arrays. Remarkably, observations based on such polarity argue against a recognition of the spacer precursors on transcript RNA molecules as a general rule.
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Barrangou, Rodolphe, Anne-Claire Coûté-Monvoisin, Buffy Stahl, Isabelle Chavichvily, Florian Damange, Dennis A. Romero, Patrick Boyaval, Christophe Fremaux und Philippe Horvath. „Genomic impact of CRISPR immunization against bacteriophages“. Biochemical Society Transactions 41, Nr. 6 (20.11.2013): 1383–91. http://dx.doi.org/10.1042/bst20130160.
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CRISPR (clustered regularly interspaced short palindromic repeats) together with cas (CRISPR-associated) genes form the CRISPR–Cas immune system, which provides sequence-specific adaptive immunity against foreign genetic elements in bacteria and archaea. Immunity is acquired by the integration of short stretches of invasive DNA as novel ‘spacers’ into CRISPR loci. Subsequently, these immune markers are transcribed and generate small non-coding interfering RNAs that specifically guide nucleases for sequence-specific cleavage of complementary sequences. Among the four CRISPR–Cas systems present in Streptococcus thermophilus, CRISPR1 and CRISPR3 have the ability to readily acquire new spacers following bacteriophage or plasmid exposure. In order to investigate the impact of building CRISPR-encoded immunity on the host chromosome, we determined the genome sequence of a BIM (bacteriophage-insensitive mutant) derived from the DGCC7710 model organism, after four consecutive rounds of bacteriophage challenge. As expected, active CRISPR loci evolved via polarized addition of several novel spacers following exposure to bacteriophages. Although analysis of the draft genome sequence revealed a variety of SNPs (single nucleotide polymorphisms) and INDELs (insertions/deletions), most of the in silico differences were not validated by Sanger re-sequencing. In addition, two SNPs and two small INDELs were identified and tracked in the intermediate variants. Overall, building CRISPR-encoded immunity does not significantly affect the genome, which allows the maintenance of important functional properties in isogenic CRISPR mutants. This is critical for the development and formulation of sustainable and robust next-generation starter cultures with increased industrial lifespans.
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Kiro, Ruth, Moran G. Goren, Ido Yosef und Udi Qimron. „CRISPR adaptation in Escherichia coli subtypeI-E system“. Biochemical Society Transactions 41, Nr. 6 (20.11.2013): 1412–15. http://dx.doi.org/10.1042/bst20130109.
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The CRISPRs (clustered regularly interspaced short palindromic repeats) and their associated Cas (CRISPR-associated) proteins are a prokaryotic adaptive defence system against foreign nucleic acids. The CRISPR array comprises short repeats flanking short segments, called ‘spacers’, which are derived from foreign nucleic acids. The process of spacer insertion into the CRISPR array is termed ‘adaptation’. Adaptation allows the system to rapidly evolve against emerging threats. In the present article, we review the most recent studies on the adaptation process, and focus primarily on the subtype I-E CRISPR–Cas system of Escherichia coli.
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Briner, Alexandra E., und Rodolphe Barrangou. „Lactobacillus buchneri Genotyping on the Basis of Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) Locus Diversity“. Applied and Environmental Microbiology 80, Nr. 3 (22.11.2013): 994–1001. http://dx.doi.org/10.1128/aem.03015-13.
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ABSTRACTClustered regularly interspaced short palindromic repeats (CRISPR) in combination with associated sequences (cas) constitute the CRISPR-Cas immune system, which uptakes DNA from invasive genetic elements as novel “spacers” that provide a genetic record of immunization events. We investigated the potential of CRISPR-based genotyping ofLactobacillus buchneri, a species relevant for commercial silage, bioethanol, and vegetable fermentations. Upon investigating the occurrence and diversity of CRISPR-Cas systems inLactobacillus buchnerigenomes, we observed a ubiquitous occurrence of CRISPR arrays containing a 36-nucleotide (nt) type II-A CRISPR locus adjacent to fourcasgenes, including the universalcas1andcas2genes and the type II signature genecas9. Comparative analysis of CRISPR spacer content in 26L. buchneripickle fermentation isolates associated with spoilage revealed 10 unique locus genotypes that contained between 9 and 29 variable spacers. We observed a set of conserved spacers at the ancestral end, reflecting a common origin, as well as leader-end polymorphisms, reflecting recent divergence. Some of these spacers showed perfect identity with phage sequences, and many spacers showed homology toLactobacillusplasmid sequences. Following a comparative analysis of sequences immediately flanking protospacers that matched CRISPR spacers, we identified a novel putative protospacer-adjacent motif (PAM), 5′-AAAA-3′. Overall, these findings suggest that type II-A CRISPR-Cas systems are valuable for genotyping ofL. buchneri.
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Silva, Adrianne M. A., Ana C. O. Luz, Keyla V. M. Xavier, Maria P. S. Barros, Hirisleide B. Alves, Marcus V. A. Batista und Tereza C. Leal-Balbino. „Analysis of CRISPR/Cas Genetic Structure, Spacer Content and Molecular Epidemiology in Brazilian Acinetobacter baumannii Clinical Isolates“. Pathogens 12, Nr. 6 (26.05.2023): 764. http://dx.doi.org/10.3390/pathogens12060764.
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CRISPR/Cas is a molecular mechanism to prevent predatory viruses from invading bacteria via the insertion of small viral sequences (spacers) in its repetitive locus. The nature of spacer incorporation and the viral origins of spacers provide an overview of the genetic evolution of bacteria, their natural viral predators, and the mechanisms that prokaryotes may use to protect themselves, or to acquire mobile genetic elements such as plasmids. Here, we report on the CRISPR/Cas genetic structure, its spacer content, and strain epidemiology through MLST and CRISPR typing in Acinetobacter baumannii, an opportunistic pathogen intimately related to hospital infections and antimicrobial resistance. Results show distinct genetic characteristics, such as polymorphisms specific to ancestor direct repeats, a well-defined degenerate repeat, and a conserved leader sequence, as well as showing most spacers as targeting bacteriophages, and several self-targeting spacers, directed at prophages. There was a particular relationship between CRISPR/Cas and CC113 in the study of Brazilian isolates, and CRISPR-related typing techniques are interesting for subtyping strains with the same MLST profile. We want to emphasize the significance of descriptive genetic research on CRISPR loci, and we argue that spacer or CRISPR typing are helpful for small-scale investigations, preferably in conjunction with other molecular typing techniques such as MLST.
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Watson, B. N. J., R. A. Easingwood, B. Tong, M. Wolf, G. P. C. Salmond, R. H. J. Staals, M. Bostina und P. C. Fineran. „Different genetic and morphological outcomes for phages targeted by single or multiple CRISPR-Cas spacers“. Philosophical Transactions of the Royal Society B: Biological Sciences 374, Nr. 1772 (25.03.2019): 20180090. http://dx.doi.org/10.1098/rstb.2018.0090.
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CRISPR-Cas systems provide bacteria and archaea with adaptive immunity against genetic invaders, such as bacteriophages. The systems integrate short sequences from the phage genome into the bacterial CRISPR array. These ‘spacers’ provide sequence-specific immunity but drive natural selection of evolved phage mutants that escape the CRISPR-Cas defence. Spacer acquisition occurs by either naive or primed adaptation. Naive adaptation typically results in the incorporation of a single spacer. By contrast, priming is a positive feedback loop that often results in acquisition of multiple spacers, which occurs when a pre-existing spacer matches the invading phage. We predicted that single and multiple spacers, representative of naive and primed adaptation, respectively, would cause differing outcomes after phage infection. We investigated the response of two phages, ϕTE and ϕM1, to the Pectobacterium atrosepticum type I-F CRISPR-Cas system and observed that escape from single spacers typically occurred via point mutations. Alternatively, phages escaped multiple spacers through deletions, which can occur in genes encoding structural proteins. Cryo-EM analysis of the ϕTE structure revealed shortened tails in escape mutants with tape measure protein deletions. We conclude that CRISPR-Cas systems can drive phage genetic diversity, altering morphology and fitness, through selective pressures arising from naive and primed acquisition events. This article is part of a discussion meeting issue ‘The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems’.
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Kuno, Sotaro, Takashi Yoshida, Takakazu Kaneko und Yoshihiko Sako. „Intricate Interactions between the Bloom-Forming Cyanobacterium Microcystis aeruginosa and Foreign Genetic Elements, Revealed by Diversified Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) Signatures“. Applied and Environmental Microbiology 78, Nr. 15 (25.05.2012): 5353–60. http://dx.doi.org/10.1128/aem.00626-12.
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ABSTRACTClustered regularly interspaced short palindromic repeats (CRISPR) confer sequence-dependent, adaptive resistance in prokaryotes against viruses and plasmids via incorporation of short sequences, called spacers, derived from foreign genetic elements. CRISPR loci are thus considered to provide records of past infections. To describe the host-parasite (i.e., cyanophages and plasmids) interactions involving the bloom-forming freshwater cyanobacteriumMicrocystis aeruginosa, we investigated CRISPR in fourM. aeruginosastrains and in two previously sequenced genomes. The number of spacers in each locus was larger than the average among prokaryotes. All spacers were strain specific, except for a string of 11 spacers shared in two closely related strains, suggesting diversification of the loci. Using CRISPR repeat-based PCR, 24 CRISPR genotypes were identified in a natural cyanobacterial community. Among 995 unique spacers obtained, only 10 sequences showed similarity toM. aeruginosaphage Ma-LMM01. Of these, six spacers showed only silent or conservative nucleotide mutations compared to Ma-LMM01 sequences, suggesting a strategy by the cyanophage to avert CRISPR immunity dependent on nucleotide identity. These results imply that host-phage interactions can be divided intoM. aeruginosa-cyanophage combinations rather than pandemics of population-wide infectious cyanophages. Spacer similarity also showed frequent exposure ofM. aeruginosato small cryptic plasmids that were observed only in a few strains. Thus, the diversification of CRISPR implies thatM. aeruginosahas been challenged by diverse communities (almost entirely uncharacterized) of cyanophages and plasmids.
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Stepanenko, L. A., Yu P. Dzhioev, V. I. Zlobin, A. Yu Borisenko, V. P. Salovarova, N. A. Arefieva, I. Zh Seminsky und I. V. Malov. „Development of screening approaches of highly specific bacteriophages based on bioinformatic analysis of CRISPR-Cas structures of Corynebacterium diphtheriae systems“. Proceedings of Universities. Applied Chemistry and Biotechnology 11, Nr. 2 (04.07.2021): 216–27. http://dx.doi.org/10.21285/2227-2925-2021-11-2-216-227.
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This study aims to develop approaches for screening highly specific bacteriophages based on bio-informatic analysis of CRISPR-Cas structures of bacterial systems using the example of Corynebacterium diphtheriae. We proposed an algorithm for bioinformatic search and analysis of CRISPR-Cas structures of bacteria systems and phage screening through spacer sequences of CRISPR-cassette in genomes of Corynebacterium strains. 22 genome-wide sequences loaded from the GenBank database were selected as the target. 21 strains out of 22 had CRISPR-Cas systems. Using several search algorithms in CRISPR-Cas systems, one CRISPR-cassette was found in 23.8% of the tested strains and two in 76.2% of cases. Near the cassettes, a complete set of Cas-genes was identified, characteristic of two types of systems: Type-I Subtype-I-E and Type-II Subtype-II-C. The conducted analysis of the CRISPR-cassette spacer composition showed 3 to 42 spacers in the cassette. The cumulative total number of identified spacers amounted to 297, 64 spacers of which repeated in two or more CRISPR-cassettes, 159 spacers had no replicates. The three pairs of strains under study from this group had a complete match of spacer and consensus sequences, although they were isolated at different times and in multiple countries. A phylogenetic analysis was performed to confirm their common origin. Phages screening through the spacer sequences showed the highest compliance of the spacers with the phages protospacers, characteristic of the bacteria of the Mycobacteriaceae, Gordoniaceae, Streptomycetaceae, Corynebacteriaceae family belonging to the Actinobacteria type. One strain with multiple antibiotic resistance was identified, and its expected bacteriophage resistance was determined using this method. Thus, the developed bioinformatic analysis technology allowed the information on the expected resistance of the tested strains CRISPR-Cas system against the detected phages to be obtained, which in the long term enables the development of a platform of personalised bacteriophage treatment approaches.
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McKitterick, Amelia C., Kristen N. LeGault, Angus Angermeyer, Munirul Alam und Kimberley D. Seed. „Competition between mobile genetic elements drives optimization of a phage-encoded CRISPR-Cas system: insights from a natural arms race“. Philosophical Transactions of the Royal Society B: Biological Sciences 374, Nr. 1772 (25.03.2019): 20180089. http://dx.doi.org/10.1098/rstb.2018.0089.
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CRISPR-Cas systems function as adaptive immune systems by acquiring nucleotide sequences called spacers that mediate sequence-specific defence against competitors. Uniquely, the phage ICP1 encodes a Type I-F CRISPR-Cas system that is deployed to target and overcome PLE, a mobile genetic element with anti-phage activity in Vibrio cholerae . Here, we exploit the arms race between ICP1 and PLE to examine spacer acquisition and interference under laboratory conditions to reconcile findings from wild populations. Natural ICP1 isolates encode multiple spacers directed against PLE, but we find that single spacers do not interfere equally with PLE mobilization. High-throughput sequencing to assay spacer acquisition reveals that ICP1 can also acquire spacers that target the V. cholerae chromosome. We find that targeting the V. cholerae chromosome proximal to PLE is sufficient to block PLE and is dependent on Cas2-3 helicase activity. We propose a model in which indirect chromosomal spacers are able to circumvent PLE by Cas2-3-mediated processive degradation of the V. cholerae chromosome before PLE mobilization. Generally, laboratory-acquired spacers are much more diverse than the subset of spacers maintained by ICP1 in nature, showing how evolutionary pressures can constrain CRISPR-Cas targeting in ways that are often not appreciated through in vitro analyses. This article is part of a discussion meeting issue ‘The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems’.
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Semenova, Ekaterina, Ekaterina Savitskaya, Olga Musharova, Alexandra Strotskaya, Daria Vorontsova, Kirill A. Datsenko, Maria D. Logacheva und Konstantin Severinov. „Highly efficient primed spacer acquisition from targets destroyed by the Escherichia coli type I-E CRISPR-Cas interfering complex“. Proceedings of the National Academy of Sciences 113, Nr. 27 (20.06.2016): 7626–31. http://dx.doi.org/10.1073/pnas.1602639113.
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Prokaryotic clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR associated (Cas) immunity relies on adaptive acquisition of spacers—short fragments of foreign DNA. For the type I-E CRISPR-Cas system from Escherichia coli, efficient “primed” adaptation requires Cas effector proteins and a CRISPR RNA (crRNA) whose spacer partially matches a segment (protospacer) in target DNA. Primed adaptation leads to selective acquisition of additional spacers from DNA molecules recognized by the effector–crRNA complex. When the crRNA spacer fully matches a protospacer, CRISPR interference—that is, target destruction without acquisition of additional spacers—is observed. We show here that when the rate of degradation of DNA with fully and partially matching crRNA targets is made equal, fully matching protospacers stimulate primed adaptation much more efficiently than partially matching ones. The result indicates that different functional outcomes of CRISPR-Cas response to two kinds of protospacers are not caused by different structures formed by the effector–crRNA complex but are due to the more rapid destruction of targets with fully matching protospacers.
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Stepanenko, L. A., B. G. Sukhov, V. V. Bedinskaya, A. Yu Borisenko und T. V. Kon’kova. „Developing approaches for search and analysis of CRISPR-Cas systems on the example of <i>Klebsiella pneumoniae</i> strains as a basis for creating personalized bacteriophage therapy“. Proceedings of Universities. Applied Chemistry and Biotechnology 13, Nr. 2 (02.07.2023): 197–205. http://dx.doi.org/10.21285/2227-2925-2023-13-2-197-205.
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This paper proposes an algorithm for searching and analyzing the structures of CRISPR-Cas systems of bacteria and screening bacteriophages through spacers in CRISPR cassettes using bioinformatic research methods in the genomes of Klebsiella pneumoniae strains. The aim was to determine and study the structure of CRISPR-Cas systems of bacteria on the example of Klebsiella pneumoniae strains using bioinformatic research methods in order to develop approaches for the selection of target bacteriophages. The research object included 150 genome-wide sequences downloaded from the GenBank database. Of these sequences, CRISPR-Cas systems were detected in 52 strains, which amounted to 34.7%. Using several search algorithms in the CRISPR-Cas systems of the studied strains, the presence of one and two CRISPR cassette was determined in 46.2 and 53.8% of cases, respectively. In all the cases, a complete set of Cas genes characteristic of Type-I Subtype-I-E systems was identified next to the cassettes. The total number of the identified spacers was 1659, of which 281 spacers were repeated in two or more CRISPR loci, while 505 spacers had no repeats. The number of spacers in the cassettes ranged from 4 to 64. The analysis of the spacer composition in CRISPR cassettes of antibiotic-resistant and hospital strains provided information on their evolutionary history and on the bacteriophages which are targeted by their CRISPR systems. The developed bioinformatic analysis algorithm enables creating a platform for the development of personalized bacteriophage therapy technologies.
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Maniv, Inbal, Wenyan Jiang, David Bikard und Luciano A. Marraffini. „Impact of Different Target Sequences on Type III CRISPR-Cas Immunity“. Journal of Bacteriology 198, Nr. 6 (11.01.2016): 941–50. http://dx.doi.org/10.1128/jb.00897-15.
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ABSTRACTClustered regularly interspaced short palindromic repeat (CRISPR) loci encode an adaptive immune system of prokaryotes. Within these loci, sequences intercalated between repeats known as “spacers” specify the targets of CRISPR immunity. The majority of spacers match sequences present in phages and plasmids; however, it is not known whether there are differences in the immunity provided against these diverse invaders. We studied this issue using theStaphylococcus epidermidisCRISPR system, which harbors spacers matching both phages and plasmids. We determined that this CRISPR system provides similar levels of defense against the conjugative plasmid pG0400 and the bacteriophage CNPX. However, whereas antiplasmid immunity was very sensitive to the introduction of mismatches in the target sequence, mutations in the phage target were largely tolerated. Placing the phage and plasmid targets into a vector that can be both conjugated and transduced, we demonstrated that the route of entry of the target has no impact on the effect of the mismatches on immunity. Instead, we established that the specific sequences of each spacer/target determine the susceptibility of theS. epidermidisCRISPR system to mutations. Therefore, spacers that are more resistant to mismatches would provide long-term immunity against phages and plasmids that otherwise would escape CRISPR targeting through the accumulation of mutations in the target sequence. These results uncover an unexpected complexity in the arms race between CRISPR-Cas systems and prokaryotic infectious genetic elements.IMPORTANCECRISPR-Cas loci protect bacteria and archaea from both phage infection and plasmid invasion. These loci harbor short sequences of phage and plasmid origin known as “spacers” that specify the targets of CRISPR-Cas immunity. The presence of a spacer sequence matching a phage or plasmid ensures host immunity against infection by these genetic elements. In turn, phages and plasmids constantly mutate their targets to avoid recognition by the spacers of the CRISPR-Cas immune system. In this study, we demonstrated that different spacer sequences vary in their ability to tolerate target mutations that allow phages and plasmids to escape from CRISPR-Cas immunity. These results uncover an unexpected complexity in the arms race between CRISPR-Cas systems and prokaryotic infectious genetic elements.
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Nussenzweig, Philip M., und Luciano A. Marraffini. „Molecular Mechanisms of CRISPR-Cas Immunity in Bacteria“. Annual Review of Genetics 54, Nr. 1 (23.11.2020): 93–120. http://dx.doi.org/10.1146/annurev-genet-022120-112523.
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Prokaryotes have developed numerous defense strategies to combat the constant threat posed by the diverse genetic parasites that endanger them. Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas loci guard their hosts with an adaptive immune system against foreign nucleic acids. Protection starts with an immunization phase, in which short pieces of the invader's genome, known as spacers, are captured and integrated into the CRISPR locus after infection. Next, during the targeting phase, spacers are transcribed into CRISPR RNAs (crRNAs) that guide CRISPR-associated (Cas) nucleases to destroy the invader's DNA or RNA. Here we describe the many different molecular mechanisms of CRISPR targeting and how they are interconnected with the immunization phase through a third phase of the CRISPR-Cas immune response: primed spacer acquisition. In this phase, Cas proteins direct the crRNA-guided acquisition of additional spacers to achieve a more rapid and robust immunization of the population.
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Kuno, Sotaro, Yoshihiko Sako und Takashi Yoshida. „Diversification of CRISPR within coexisting genotypes in a natural population of the bloom-forming cyanobacterium Microcystis aeruginosa“. Microbiology 160, Nr. 5 (01.05.2014): 903–16. http://dx.doi.org/10.1099/mic.0.073494-0.
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The clustered regularly interspaced short palindromic repeat (CRISPR) confers adaptive immunity against phages via sequence fragments (spacers) derived from mobile genetic elements (MGEs), thus serving as a memory of past host–phage co-evolution. To understand co-evolutionary dynamics in natural settings, we examined CRISPR diversity in 94 isolates of Microcystis aeruginosa from a small eutrophic pond. Fifty-two isolates possessed the CRISPR and were classified into 22 different CRISPR-related genotypes, suggesting stable coexistence of multiple genotypes with different phage susceptibility. Seven CRISPR-related genotypes showed variation of spacers at the leader-end of the CRISPR, indicating active spacer addition from MGEs. An abundant phylotype (based on the internal transcribed spacer of the rRNA gene) contained different CRISPR spacer genotypes with the same CRISPR-associated cas2 gene. These data suggest that selective phage infection and possibly plasmid transfer may contribute to maintenance of multiple genotypes of M. aeruginosa and that rapid co-evolution within a host–phage combination may be driven by increased contact frequency. Forty-two isolates lacked detectable CRISPR loci. Relative abundance of the CRISPR-lacking genotypes in the population suggests that CRISPR loss may be selected for enhanced genetic exchange.
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Shiriaeva, Anna, Ivan Fedorov, Danylo Vyhovskyi und Konstantin Severinov. „Detection of CRISPR adaptation“. Biochemical Society Transactions 48, Nr. 1 (03.02.2020): 257–69. http://dx.doi.org/10.1042/bst20190662.
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Prokaryotic adaptive immunity is built when short DNA fragments called spacers are acquired into CRISPR (clustered regularly interspaced short palindromic repeats) arrays. CRISPR adaptation is a multistep process which comprises selection, generation, and incorporation of prespacers into arrays. Once adapted, spacers provide immunity through the recognition of complementary nucleic acid sequences, channeling them for destruction. To prevent deleterious autoimmunity, CRISPR adaptation must therefore be a highly regulated and infrequent process, at least in the absence of genetic invaders. Over the years, ingenious methods to study CRISPR adaptation have been developed. In this paper, we discuss and compare methods that detect CRISPR adaptation and its intermediates in vivo and propose suppressing PCR as a simple modification of a popular assay to monitor spacer acquisition with increased sensitivity.
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González-Delgado, Alejandro, Mario Rodríguez Mestre, Francisco Martínez-Abarca und Nicolás Toro. „Spacer acquisition from RNA mediated by a natural reverse transcriptase-Cas1 fusion protein associated with a type III-D CRISPR–Cas system in Vibrio vulnificus“. Nucleic Acids Research 47, Nr. 19 (04.09.2019): 10202–11. http://dx.doi.org/10.1093/nar/gkz746.
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Abstract The association of reverse transcriptases (RTs) with CRISPR–Cas system has recently attracted interest because the RT activity appears to facilitate the RT-dependent acquisition of spacers from RNA molecules. However, our understanding of this spacer acquisition process remains limited. We characterized the in vivo acquisition of spacers mediated by an RT-Cas1 fusion protein linked to a type III-D system from Vibrio vulnificus strain YJ016, and showed that the adaptation module, consisting of the RT-Cas1 fusion, two different Cas2 proteins (A and B) and one of the two CRISPR arrays, was completely functional in a heterologous host. We found that mutations of the active site of the RT domain significantly decreased the acquisition of new spacers and showed that this RT-Cas1-associated adaptation module was able to incorporate spacers from RNA molecules into the CRISPR array. We demonstrated that the two Cas2 proteins of the adaptation module were required for spacer acquisition. Furthermore, we found that several sequence-specific features were required for the acquisition and integration of spacers derived from any region of the genome, with no bias along the 5′and 3′ends of coding sequences. This study provides new insight into the RT-Cas1 fusion protein-mediated acquisition of spacers from RNA molecules.
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Hsu, Jen-Fu, Jang-Jih Lu, Chih Lin, Shih-Ming Chu, Lee-Chung Lin, Mei-Yin Lai, Hsuan-Rong Huang, Ming-Chou Chiang und Ming-Horng Tsai. „Clustered Regularly Interspaced Short Palindromic Repeat Analysis of Clonal Complex 17 Serotype III Group B Streptococcus Strains Causing Neonatal Invasive Diseases“. International Journal of Molecular Sciences 22, Nr. 21 (27.10.2021): 11626. http://dx.doi.org/10.3390/ijms222111626.
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Group B Streptococcus (GBS) is an important pathogen of neonatal infections, and the clonal complex (CC)-17/serotype III GBS strain has emerged as the dominant strain. The clinical manifestations of CC17/III GBS sepsis may vary greatly but have not been well-investigated. A total of 103 CC17/III GBS isolates that caused neonatal invasive diseases were studied using a new approach based on clustered regularly interspaced short palindromic repeats (CRISPR) loci and restriction fragment length polymorphism (RFLP) analyses. All spacers of CRISPR loci were sequenced and analyzed with the clinical presentations. After CRISPR-RFLP analyses, a total of 11 different patterns were observed among the 103 CRISPR-positive GBS isolates. GBS isolates with the same RFLP patterns were found to have highly comparable spacer contents. Comparative sequence analysis of the CRISPR1 spacer content revealed that it is highly diverse and consistent with the dynamics of this system. A total of 29 of 43 (67.4%) spacers displayed homology to reported phage and plasmid DNA sequences. In addition, all CC17/III GBS isolates could be categorized into three subgroups based on the CRISPR-RFLP patterns and eBURST analysis. The CC17/III GBS isolates with a specific CRISPR-RFLP pattern were more significantly associated with occurrences of severe sepsis (57.1% vs. 29.3%, p = 0.012) and meningitis (50.0% vs. 20.8%, p = 0.009) than GBS isolates with RFLP lengths between 1000 and 1300 bp. Whole-genome sequencing was also performed to verify the differences between CC17/III GBS isolates with different CRISPR-RFLP patterns. We concluded that the CRISPR-RFLP analysis is potentially applicable to categorizing CC17/III GBS isolates, and a specific CRISPR-RFLP pattern could be used as a new biomarker to predict meningitis and illness severity after further verification.
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Kurilovich, Elena, Anna Shiriaeva, Anastasia Metlitskaya, Natalia Morozova, Ivana Ivancic-Bace, Konstantin Severinov und Ekaterina Savitskaya. „Genome Maintenance Proteins Modulate Autoimmunity Mediated Primed Adaptation by the Escherichia coli Type I-E CRISPR-Cas System“. Genes 10, Nr. 11 (31.10.2019): 872. http://dx.doi.org/10.3390/genes10110872.
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Bacteria and archaea use CRISPR-Cas adaptive immunity systems to interfere with viruses, plasmids, and other mobile genetic elements. During the process of adaptation, CRISPR-Cas systems acquire immunity by incorporating short fragments of invaders’ genomes into CRISPR arrays. The acquisition of fragments of host genomes leads to autoimmunity and may drive chromosomal rearrangements, negative cell selection, and influence bacterial evolution. In this study, we investigated the role of proteins involved in genome stability maintenance in spacer acquisition by the Escherichia coli type I-E CRISPR-Cas system targeting its own genome. We show here, that the deletion of recJ decreases adaptation efficiency and affects accuracy of spacers incorporation into CRISPR array. Primed adaptation efficiency is also dramatically inhibited in double mutants lacking recB and sbcD but not in single mutants suggesting independent involvement and redundancy of RecBCD and SbcCD pathways in spacer acquisition. While the presence of at least one of two complexes is crucial for efficient primed adaptation, RecBCD and SbcCD affect the pattern of acquired spacers. Overall, our data suggest distinct roles of the RecBCD and SbcCD complexes and of RecJ in spacer precursor selection and insertion into CRISPR array and highlight the functional interplay between CRISPR-Cas systems and host genome maintenance mechanisms.
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Moller, Abraham G., und Chun Liang. „MetaCRAST: reference-guided extraction of CRISPR spacers from unassembled metagenomes“. PeerJ 5 (07.09.2017): e3788. http://dx.doi.org/10.7717/peerj.3788.
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Clustered regularly interspaced short palindromic repeat (CRISPR) systems are the adaptive immune systems of bacteria and archaea against viral infection. While CRISPRs have been exploited as a tool for genetic engineering, their spacer sequences can also provide valuable insights into microbial ecology by linking environmental viruses to their microbial hosts. Despite this importance, metagenomic CRISPR detection remains a major challenge. Here we present a reference-guided CRISPR spacer detection tool (Metagenomic CRISPR Reference-Aided Search Tool—MetaCRAST) that constrains searches based on user-specified direct repeats (DRs). These DRs could be expected from assembly or taxonomic profiles of metagenomes. We compared the performance of MetaCRAST to those of two existing metagenomic CRISPR detection tools—Crass and MinCED—using both real and simulated acid mine drainage (AMD) and enhanced biological phosphorus removal (EBPR) metagenomes. Our evaluation shows MetaCRAST improves CRISPR spacer detection in real metagenomes compared to the de novo CRISPR detection methods Crass and MinCED. Evaluation on simulated metagenomes show it performs better than de novo tools for Illumina metagenomes and comparably for 454 metagenomes. It also has comparable performance dependence on read length and community composition, run time, and accuracy to these tools. MetaCRAST is implemented in Perl, parallelizable through the Many Core Engine (MCE), and takes metagenomic sequence reads and direct repeat queries (FASTA or FASTQ) as input. It is freely available for download at https://github.com/molleraj/MetaCRAST.
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Pourcel, C., G. Salvignol und G. Vergnaud. „CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies“. Microbiology 151, Nr. 3 (01.03.2005): 653–63. http://dx.doi.org/10.1099/mic.0.27437-0.
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The remarkable repetitive elements called CRISPRs (clustered regularly interspaced short palindromic repeats) consist of repeats interspaced with non-repetitive elements or ‘spacers’. CRISPRs are present in both archaea and bacteria, in association with genes involved in DNA recombination and repair. In the Yersinia pestis genome, three such elements are found at three distinct loci, one of them being highly polymorphic. The authors have sequenced a total of 109 alleles of the three Y. pestis CRISPRs and they describe 29 new spacers, most being specific to one isolate. In nine strains of Yersinia pseudotuberculosis, 132 spacers were found, of which only three are common to Y. pestis isolates. In Y. pestis of the Orientalis biovar investigated in detail here, deletion of motifs is observed but it appears that addition of new motifs to a common ancestral element is the most frequent event. This takes place at the three different loci, although at a higher rate in one of the loci, and the addition of new motifs is polarized. Interestingly, the most recently acquired spacers were found to have a homologue at another locus in the genome, the majority of these inside an inactive prophage. This is believed to be the first time that the origin of the spacers in CRISPR elements has been explained. The CRISPR structure provides a new and robust identification tool.
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Aviram, Naama, Ashley N. Thornal, David Zeevi und Luciano A. Marraffini. „Different modes of spacer acquisition by the Staphylococcus epidermidis type III-A CRISPR-Cas system“. Nucleic Acids Research 50, Nr. 3 (20.01.2022): 1661–72. http://dx.doi.org/10.1093/nar/gkab1299.
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Abstract CRISPR-Cas systems provide prokaryotic organisms with an adaptive defense mechanism that acquires immunological memories of infections. This is accomplished by integration of short fragments from the genome of invaders such as phages and plasmids, called ‘spacers’, into the CRISPR locus of the host. Depending on their genetic composition, CRISPR-Cas systems can be classified into six types, I-VI, however spacer acquisition has been extensively studied only in type I and II systems. Here, we used an inducible spacer acquisition assay to study this process in the type III-A CRISPR-Cas system of Staphylococcus epidermidis, in the absence of phage selection. Similarly to type I and II spacer acquisition, this type III system uses Cas1 and Cas2 to preferentially integrate spacers from the chromosomal terminus and free dsDNA ends produced after DNA breaks, in a manner that is enhanced by the AddAB DNA repair complex. Surprisingly, a different mode of spacer acquisition from rRNA and tRNA loci, which spans only the transcribed sequences of these genes and is not enhanced by AddAB, was also detected. Therefore, our findings reveal both common mechanistic principles that may be conserved in all CRISPR-Cas systems, as well as unique and intriguing features of type III spacer acquisition.
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Garrett, Sandra, Masami Shiimori, Elizabeth A. Watts, Landon Clark, Brenton R. Graveley und Michael P. Terns. „Primed CRISPR DNA uptake in Pyrococcus furiosus“. Nucleic Acids Research 48, Nr. 11 (18.05.2020): 6120–35. http://dx.doi.org/10.1093/nar/gkaa381.
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Abstract CRISPR-Cas adaptive immune systems are used by prokaryotes to defend against invaders like viruses and other mobile genetic elements. Immune memories are stored in the form of ‘spacers’ which are short DNA sequences that are captured from invaders and added to the CRISPR array during a process called ‘adaptation’. Spacers are transcribed and the resulting CRISPR (cr)RNAs assemble with different Cas proteins to form effector complexes that recognize matching nucleic acid and destroy it (‘interference’). Adaptation can be ‘naïve’, i.e. independent of any existing spacer matches, or it can be ‘primed’, i.e. spurred by the crRNA-mediated detection of a complete or partial match to an invader sequence. Here we show that primed adaptation occurs in Pyrococcus furiosus. Although P. furiosus has three distinct CRISPR-Cas interference systems (I-B, I-A and III-B), only the I-B system and Cas3 were necessary for priming. Cas4, which is important for selection and processing of new spacers in naïve adaptation, was also essential for priming. Loss of either the I-B effector proteins or Cas3 reduced naïve adaptation. However, when Cas3 and all crRNP genes were deleted, uptake of correctly processed spacers was observed, indicating that none of these interference proteins are necessary for naïve adaptation.
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Bedinskaya, V. V., L. A. Stepanenko, E. V. Simonova, A. G. Atlas, E. B. Rakova und V. I. Zlobin. „Characterization of CRISPR/CAS System in Pseudomonas aeruginosa DSM 50071 Based on Bioinformatic Analysis of its Structures“. Bulletin of Irkutsk State University. Series Biology. Ecology 40 (2022): 3–14. http://dx.doi.org/10.26516/2073-3372.2022.40.3.
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An algorithm for bioinformatic search and analysis of the structures of CRISPR/Cas systems of bacteria and screening of phages and plasmids through spacer sequences of CRISPR cassettes in the genome of the Pseudomonas aeruginosa strain DSM 50071 is presented.Using several search algorithms in the CRISPR/Cas system of the studied strain, the presence of three CRISPR loci and a group of Cas genes characteristic of Type-I Subtype-I-F was determined.Analysis of the spacer composition of CRISPR cassettes showed the presence of 31 to 43 spacers and a universal consensus repeat in all cassettes.Screening of the spacer sequences of the CRISPR cassettes of the studied strain showed their correspondence to the protospacers of phages and plasmids of bacteria of the families Pseudomonadaceae and Enterobacteriaceae. A complete characterization of bacteriophages to which this strain is resistant is given with their accession number in NCBI. A complete identification of spacers to protospacers of phages specific for bacteria of the Pseudomonadaceae family, most often isolated from the lungs of patients with bronchiectasis, pneumonia, as well as from hospitals and reservoirs, has been established.Full correspondence between spacers and protospacers of bacterial plasmids with pan-resistance and causing the development of respiratory failure and pneumonia was revealed. Correspondence of a segment of one spacer with protospacers of several bacterial phages of the same family was noted. This may indicate that the bacterium “expediently” acquires new spacers from DNA regions that are conserved for phages of bacteria of the same family.Genes that have phage protospacers in their structure have been identified.It has been established that these genes are responsible for the synthesis of enzymes that regulate the processes of virus reproduction.Therefore, activation of the CRISPR/Cas system in the genome of this strain will allow the restriction endonuclease to introduce breaks into unmethylated DNA, which will lead to disruption of the synthesis of this enzyme, and, consequently, disruption of bacteriophage replication.Correspondences of spacer sequences with protospacers of plasmids included in the structure of genes responsible for the synthesis of conjugative transfer enzymes were revealed.These results suggested that activation of the CRISPR/Cas system of this strain would disrupt the processes replication of bacteriophage and conjugation.The proposed algorithm made it possible to obtain information about the structure of the CRISPR/Cas system of the P. aeruginosa DSM 50071 strain, about its resistance to certain phages and plasmids. In the future, this will serve as the basis for creating approaches for targeted therapy of infectious diseases.
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Borisenko, A. Yu, N. A. Arefieva, Yu P. Dzhioev, S. V. Erdyneev, Yu S. Bukin, G. A. Teterina, A. A. Pristavka et al. „In Silico Analysis of the Structural Diversity of CRISPR-Cas Systems in Genomes of Salmonella enterica and Phage Species Detected by Them“. Bulletin of Irkutsk State University. Series Biology. Ecology 45 (2023): 3–20. http://dx.doi.org/10.26516/2073-3372.2023.45.3.
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The problem of resistance of pathogenic bacteria to antibiotics has become global and, therefore, there is renewed interest in the use of bacteriophages. However, bacteria also have phage defense structures, the CRISPR/Cas system. Therefore, the analysis of the structural diversity of CRISPR-Cas systems in the genomes of pathogenic bacteria and phages is an important fundamental and applied direction. The aim. Investigation of the diversity of structures of CRISPR/Cas systems in the genomes of S. enterica strains from the NCBI database using bioinformatics programs and assessment of the possibilities to identify phage protection of strains through spacers in CRISPR cassettes. The studies were carried out with the genomes of 449 S. enterica strains from the NCBI database. A number of bioinformation software methods were used: 1) MacSyFinder, 2) CRISPR Interactive database, 3) CRISPR R Tool, 4) CRISPI: a CRISPR Interactive database, 5) CRISPRFinder. Screening of phages through spacers CRISPR cassettes was used: 1) CRISPRTarget, 2) Mycobacteriophage Database, 3) Phages database. In the genomes of the studied strains of S. enterica, one type of CRISPR/Cas system, I-E, was identified. Protein genes were present in each locus of the CRISPR/Cas systems: Cas1_0_I-E_7, Cas2_0_I-E_8, Cas3_0_I_1, Cas5_0_I-E_5, Cas6_0_I-E_6, Cas7_0_I-E_4, Cse1_0_I-E_2, Cse2_0_I-E_3. The number of cassettes was from 1 to 3, and the spacers in them varied from 8 to 30. Repeats in CRISPR cassettes varied from 27 to 29 base pairs. The identified phages belonged to bacteria of the genera: Salmonella – 60%, Escherichia – 18%, Enterobacter – 9%, Salmonella – 8%, and Staphylococcus and Enterococcus were up to 5%. The obtained data on the diversity of CRISPR/Cas systems in the genomes of the studied S. enterica strains demonstrate their unique structures. The homogeneity of CRISPR/Cas systems and the rooting of CAS types I-E in genomes can be explained by their participation in the interspecific transmission of these CRISPR systems.
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Bonsma-Fisher, Madeleine, Dominique Soutière und Sidhartha Goyal. „How adaptive immunity constrains the composition and fate of large bacterial populations“. Proceedings of the National Academy of Sciences 115, Nr. 32 (23.07.2018): E7462—E7468. http://dx.doi.org/10.1073/pnas.1802887115.
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Features of the CRISPR-Cas system, in which bacteria integrate small segments of phage genome (spacers) into their DNA to neutralize future attacks, suggest that its effect is not limited to individual bacteria but may control the fate and structure of whole populations. Emphasizing the population-level impact of the CRISPR-Cas system, recent experiments show that some bacteria regulate CRISPR-associated genes via the quorum sensing (QS) pathway. Here we present a model that shows that from the highly stochastic dynamics of individual spacers under QS control emerges a rank-abundance distribution of spacers that is time invariant, a surprising prediction that we test with dynamic spacer-tracking data from literature. This distribution depends on the state of the competing phage–bacteria population, which due to QS-based regulation may coexist in multiple stable states that vary significantly in their phage-to-bacterium ratio, a widely used ecological measure to characterize microbial systems.
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Sorokin, Valery A., Mikhail S. Gelfand und Irena I. Artamonova. „Evolutionary Dynamics of Clustered Irregularly Interspaced Short Palindromic Repeat Systems in the Ocean Metagenome“. Applied and Environmental Microbiology 76, Nr. 7 (29.01.2010): 2136–44. http://dx.doi.org/10.1128/aem.01985-09.
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ABSTRACT Clustered regularly interspaced short palindromic repeats (CRISPRs) form a recently characterized type of prokaryotic antiphage defense system. The phage-host interactions involving CRISPRs have been studied in experiments with selected bacterial or archaeal species and, computationally, in completely sequenced genomes. However, these studies do not allow one to take prokaryotic population diversity and phage-host interaction dynamics into account. This gap can be filled by using metagenomic data: in particular, the largest existing data set, generated from the Sorcerer II Global Ocean Sampling expedition. The application of three publicly available CRISPR recognition programs to the Global Ocean metagenome produced a large proportion of false-positive results. To address this problem, a filtering procedure was designed. It resulted in about 200 reliable CRISPR cassettes, which were then studied in detail. The repeat consensuses were clustered into several stable classes that differed from the existing classification. Short fragments of DNA similar to the cassette spacers were more frequently present in the same geographical location than in other locations (P, <0.0001). We developed a catalogue of elementary CRISPR-forming events and reconstructed the likely evolutionary history of cassettes that had common spacers. Metagenomic collections allow for relatively unbiased analysis of phage-host interactions and CRISPR evolution. The results of this study demonstrate that CRISPR cassettes retain the memory of the local virus population at a particular ocean location. CRISPR evolution may be described using a limited vocabulary of elementary events that have a natural biological interpretation.
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Deecker, Shayna R., und Alexander W. Ensminger. „Type I-F CRISPR-Cas Distribution and Array Dynamics in Legionella pneumophila“. G3: Genes|Genomes|Genetics 10, Nr. 3 (14.01.2020): 1039–50. http://dx.doi.org/10.1534/g3.119.400813.
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In bacteria and archaea, several distinct types of CRISPR-Cas systems provide adaptive immunity through broadly similar mechanisms: short nucleic acid sequences derived from foreign DNA, known as spacers, engage in complementary base pairing with invasive genetic elements setting the stage for nucleases to degrade the target DNA. A hallmark of type I CRISPR-Cas systems is their ability to acquire spacers in response to both new and previously encountered invaders (naïve and primed acquisition, respectively). Our phylogenetic analyses of 43 L. pneumophila type I-F CRISPR-Cas systems and their resident genomes suggest that many of these systems have been horizontally acquired. These systems are frequently encoded on plasmids and can co-occur with nearly identical chromosomal loci. We show that two such co-occurring systems are highly protective and undergo efficient primed acquisition in the lab. Furthermore, we observe that targeting by one system’s array can prime spacer acquisition in the other. Lastly, we provide experimental and genomic evidence for a model in which primed acquisition can efficiently replenish a depleted type I CRISPR array following a mass spacer deletion event.
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Bozic, Bojan, Jelena Repac und Marko Djordjevic. „Endogenous Gene Regulation as a Predicted Main Function of Type I-E CRISPR/Cas System in E. coli“. Molecules 24, Nr. 4 (21.02.2019): 784. http://dx.doi.org/10.3390/molecules24040784.
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CRISPR/Cas is an adaptive bacterial immune system, whose CRISPR array can actively change in response to viral infections. However, Type I-E CRISPR/Cas in E. coli (an established model system), appears not to exhibit such active adaptation, which suggests that it might have functions other than immune response. Through computational analysis, we address the involvement of the system in non-canonical functions. To assess targets of CRISPR spacers, we align them against both E. coli genome and an exhaustive (~230) set of E. coli viruses. We systematically investigate the obtained alignments, such as hit distribution with respect to genome annotation, propensity to target mRNA, the target functional enrichment, conservation of CRISPR spacers and putative targets in related bacterial genomes. We find that CRISPR spacers have a statistically highly significant tendency to target i) host compared to phage genomes, ii) one of the two DNA strands, iii) genomic dsDNA rather than mRNA, iv) transcriptionally active regions, and v) sequences (cis-regulatory elements) with slower turn-over rate compared to CRISPR spacers (trans-factors). The results suggest that the Type I-E CRISPR/Cas system has a major role in transcription regulation of endogenous genes, with a potential to rapidly rewire these regulatory interactions, with targets being selected through naïve adaptation.
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Artamonova, Daria, Karyna Karneyeva, Sofia Medvedeva, Evgeny Klimuk, Matvey Kolesnik, Anna Yasinskaya, Aleksei Samolygo und Konstantin Severinov. „Spacer acquisition by Type III CRISPR–Cas system during bacteriophage infection of Thermus thermophilus“. Nucleic Acids Research 48, Nr. 17 (21.08.2020): 9787–803. http://dx.doi.org/10.1093/nar/gkaa685.
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Abstract Type III CRISPR–Cas systems provide immunity to foreign DNA by targeting its transcripts. Target recognition activates RNases and DNases that may either destroy foreign DNA directly or elicit collateral damage inducing death of infected cells. While some Type III systems encode a reverse transcriptase to acquire spacers from foreign transcripts, most contain conventional spacer acquisition machinery found in DNA-targeting systems. We studied Type III spacer acquisition in phage-infected Thermus thermophilus, a bacterium that lacks either a standalone reverse transcriptase or its fusion to spacer integrase Cas1. Cells with spacers targeting a subset of phage transcripts survived the infection, indicating that Type III immunity does not operate through altruistic suicide. In the absence of selection spacers were acquired from both strands of phage DNA, indicating that no mechanism ensuring acquisition of RNA-targeting spacers exists. Spacers that protect the host from the phage demonstrate a very strong strand bias due to positive selection during infection. Phages that escaped Type III interference accumulated deletions of integral number of codons in an essential gene and much longer deletions in a non-essential gene. This and the fact that Type III immunity can be provided by plasmid-borne mini-arrays open ways for genomic manipulation of Thermus phages.
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Tanmoy, Arif Mohammad, Chinmoy Saha, Mohammad Saiful Islam Sajib, Senjuti Saha, Florence Komurian-Pradel, Alex van Belkum, Rogier Louwen, Samir Kumar Saha und Hubert P. Endtz. „CRISPR-Cas Diversity in Clinical Salmonella enterica Serovar Typhi Isolates from South Asian Countries“. Genes 11, Nr. 11 (18.11.2020): 1365. http://dx.doi.org/10.3390/genes11111365.
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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 >2) and B (evidence level ≤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.
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Stamereilers, Casey, Simon Wong und Philippos K. Tsourkas. „Characterization of CRISPR Spacer and Protospacer Sequences in Paenibacillus larvae and Its Bacteriophages“. Viruses 13, Nr. 3 (11.03.2021): 459. http://dx.doi.org/10.3390/v13030459.
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The bacterium Paenibacillus larvae is the causative agent of American foulbrood, the most devastating bacterial disease of honeybees. Because P. larvae is antibiotic resistant, phages that infect it are currently used as alternative treatments. However, the acquisition by P. larvae of CRISPR spacer sequences from the phages could be an obstacle to treatment efforts. We searched nine complete genomes of P. larvae strains and identified 714 CRISPR spacer sequences, of which 384 are unique. Of the four epidemiologically important P. larvae strains, three of these have fewer than 20 spacers, while one strain has over 150 spacers. Of the 384 unique spacers, 18 are found as protospacers in the genomes of 49 currently sequenced P. larvae phages. One P. larvae strain does not have any protospacers found in phages, while another has eight. Protospacer distribution in the phages is uneven, with two phages having up to four protospacers, while a third of phages have none. Some phages lack protospacers found in closely related phages due to point mutations, indicating a possible escape mechanism. This study serve a point of reference for future studies on the CRISPR-Cas system in P. larvae as well as for comparative studies of other phage–host systems.
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Nobrega, Franklin L., Hielke Walinga, Bas E. Dutilh und Stan J. J. Brouns. „Prophages are associated with extensive CRISPR–Cas auto-immunity“. Nucleic Acids Research 48, Nr. 21 (21.11.2020): 12074–84. http://dx.doi.org/10.1093/nar/gkaa1071.
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Abstract CRISPR–Cas systems require discriminating self from non-self DNA during adaptation and interference. Yet, multiple cases have been reported of bacteria containing self-targeting spacers (STS), i.e. CRISPR spacers targeting protospacers on the same genome. STS has been suggested to reflect potential auto-immunity as an unwanted side effect of CRISPR–Cas defense, or a regulatory mechanism for gene expression. Here we investigated the incidence, distribution, and evasion of STS in over 100 000 bacterial genomes. We found STS in all CRISPR–Cas types and in one fifth of all CRISPR-carrying bacteria. Notably, up to 40% of I-B and I-F CRISPR–Cas systems contained STS. We observed that STS-containing genomes almost always carry a prophage and that STS map to prophage regions in more than half of the cases. Despite carrying STS, genetic deterioration of CRISPR–Cas systems appears to be rare, suggesting a level of escape from the potentially deleterious effects of STS by other mechanisms such as anti-CRISPR proteins and CRISPR target mutations. We propose a scenario where it is common to acquire an STS against a prophage, and this may trigger more extensive STS buildup by primed spacer acquisition in type I systems, without detrimental autoimmunity effects as mechanisms of auto-immunity evasion create tolerance to STS-targeted prophages.
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Heussler, Gary E., und George A. O'Toole. „Friendly Fire: Biological Functions and Consequences of Chromosomal Targeting by CRISPR-Cas Systems“. Journal of Bacteriology 198, Nr. 10 (29.02.2016): 1481–86. http://dx.doi.org/10.1128/jb.00086-16.
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Clusteredregularlyinterspacedshortpalindromicrepeat (CRISPR)-associated (Cas) systems in bacteria and archaea target foreign elements, such as bacteriophages and conjugative plasmids, through the incorporation of short sequences (termed spacers) from the foreign element into the CRISPR array, thereby allowing sequence-specific targeting of the invader. Thus, CRISPR-Cas systems are typically considered a microbial adaptive immune system. While many of these incorporated spacers match targets on bacteriophages and plasmids, a noticeable number are derived from chromosomal DNA. While usually lethal to the self-targeting bacteria, in certain circumstances, these self-targeting spacers can have profound effects in regard to microbial biology, including functions beyond adaptive immunity. In this minireview, we discuss recent studies that focus on the functions and consequences of CRISPR-Cas self-targeting, including reshaping of the host population, group behavior modification, and the potential applications of CRISPR-Cas self-targeting as a tool in microbial biotechnology. Understanding the effects of CRISPR-Cas self-targeting is vital to fully understanding the spectrum of function of these systems.
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Wang, Kai, und Chun Liang. „CRF: detection of CRISPR arrays using random forest“. PeerJ 5 (25.04.2017): e3219. http://dx.doi.org/10.7717/peerj.3219.
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CRISPRs (clustered regularly interspaced short palindromic repeats) are particular repeat sequences found in wide range of bacteria and archaea genomes. Several tools are available for detecting CRISPR arrays in the genomes of both domains. Here we developed a new web-based CRISPR detection tool named CRF (CRISPR Finder by Random Forest). Different from other CRISPR detection tools, a random forest classifier was used in CRF to filter out invalid CRISPR arrays from all putative candidates and accordingly enhanced detection accuracy. In CRF, particularly, triplet elements that combine both sequence content and structure information were extracted from CRISPR repeats for classifier training. The classifier achieved high accuracy and sensitivity. Moreover, CRF offers a highly interactive web interface for robust data visualization that is not available among other CRISPR detection tools. After detection, the query sequence, CRISPR array architecture, and the sequences and secondary structures of CRISPR repeats and spacers can be visualized for visual examination and validation. CRF is freely available at http://bioinfolab.miamioh.edu/crf/home.php.
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Chaturvedi, Sarika, und Jinny Tomar. „CRISPR/CAS 9 Mediated Treatment for UTIs“. International Journal for Modern Trends in Science and Technology 6, Nr. 5 (31.05.2020): 82–94. http://dx.doi.org/10.46501/ijmtst060515.
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“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).
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Cady, K. C., A. S. White, J. H. Hammond, M. D. Abendroth, R. S. G. Karthikeyan, P. Lalitha, M. E. Zegans und G. A. O'Toole. „Prevalence, conservation and functional analysis of Yersinia and Escherichia CRISPR regions in clinical Pseudomonas aeruginosa isolates“. Microbiology 157, Nr. 2 (01.02.2011): 430–37. http://dx.doi.org/10.1099/mic.0.045732-0.
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Here, we report the characterization of 122 Pseudomonas aeruginosa clinical isolates from three distinct geographical locations: Dartmouth Hitchcock Medical Center in New Hampshire, USA, the Charles T. Campbell Eye Microbiology Lab at the University of Pittsburgh Medical Center, USA, and the Aravind Eye Hospital in Madurai, India. We identified and located clustered regularly interspaced short palindromic repeats (CRISPR) in 45/122 clinical isolates and sequenced these CRISPR, finding that Yersinia subtype CRISPR regions (33 %) were more prevalent than the Escherichia CRISPR region subtype (6 %) in these P. aeruginosa clinical isolates. Further, we observed 132 unique spacers from these 45 CRISPR that are 100 % identical to prophages or sequenced temperate bacteriophage capable of becoming prophages. Most intriguingly, all of these 132 viral spacers matched to temperate bacteriophage/prophages capable of inserting into the host chromosome, but not to extrachromosomally replicating lytic P. aeruginosa bacteriophage. We next assessed the ability of the more prevalent Yersinia subtype CRISPR regions to mediate resistance to bacteriophage infection or lysogeny by deleting the entire CRISPR region from sequenced strain UCBPP-PA14 and six clinical isolates. We found no change in CRISPR-mediated resistance to bacteriophage infection or lysogeny rate even for CRISPR with spacers 100 % identical to a region of the infecting bacteriophage. Lastly, to show these CRISPR and cas genes were expressed and functional, we demonstrated production of small CRISPR RNAs. This work provides both the first examination to our knowledge of CRISPR regions within clinical P. aeruginosa isolates and a collection of defined CRISPR-positive and -negative strains for further CRISPR and cas gene studies.
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Grainy, Julie, Sandra Garrett, Brenton R. Graveley und Michael P. Terns. „CRISPR repeat sequences and relative spacing specify DNA integration by Pyrococcus furiosus Cas1 and Cas2“. Nucleic Acids Research 47, Nr. 14 (20.06.2019): 7518–31. http://dx.doi.org/10.1093/nar/gkz548.
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Abstract Acquiring foreign spacer DNA into the CRISPR locus is an essential primary step of the CRISPR–Cas pathway in prokaryotes for developing host immunity to mobile genetic elements. Here, we investigate spacer integration in vitro using proteins from Pyrococcus furiosus and demonstrate that Cas1 and Cas2 are sufficient to accurately integrate spacers into a minimal CRISPR locus. Using high-throughput sequencing, we identified high frequency spacer integration occurring at the same CRISPR repeat border sites utilized in vivo, as well as at several non-CRISPR plasmid sequences which share features with repeats. Analysis of non-CRISPR integration sites revealed that Cas1 and Cas2 are directed to catalyze full-site spacer integration at specific DNA stretches where guanines and/or cytosines are 30 base pairs apart and the intervening sequence harbors several positionally conserved bases. Moreover, assaying a series of CRISPR repeat mutations, followed by sequencing of the integration products, revealed that the specificity of integration is primarily directed by sequences at the leader-repeat junction as well as an adenine-rich sequence block in the mid-repeat. Together, our results indicate that P. furiosus Cas1 and Cas2 recognize multiple sequence features distributed over a 30 base pair DNA region for accurate spacer integration at the CRISPR repeat.
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Rezzonico, Fabio, Theo H. M. Smits und Brion Duffy. „Diversity, Evolution, and Functionality of Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) Regions in the Fire Blight Pathogen Erwinia amylovora“. Applied and Environmental Microbiology 77, Nr. 11 (01.04.2011): 3819–29. http://dx.doi.org/10.1128/aem.00177-11.
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ABSTRACTThe clustered regularly interspaced short palindromic repeat (CRISPR)/Cas system confers acquired heritable immunity against mobile nucleic acid elements in prokaryotes, limiting phage infection and horizontal gene transfer of plasmids. In CRISPR arrays, characteristic repeats are interspersed with similarly sized nonrepetitive spacers derived from transmissible genetic elements and acquired when the cell is challenged with foreign DNA. New spacers are added sequentially and the number and type of CRISPR units can differ among strains, providing a record of phage/plasmid exposure within a species and giving a valuable typing tool. The aim of this work was to investigate CRISPR diversity in the highly homogeneous speciesErwinia amylovora, the causal agent of fire blight. A total of 18 CRISPR genotypes were defined within a collection of 37 cosmopolitan strains. Strains from Spiraeoideae plants clustered in three major groups: groups II and III were composed exclusively of bacteria originating from the United States, whereas group I generally contained strains of more recent dissemination obtained in Europe, New Zealand, and the Middle East. Strains from Rosoideae and Indian hawthorn (Rhaphiolepis indica) clustered separately and displayed a higher intrinsic diversity than that of isolates from Spiraeoideae plants. Reciprocal exclusion was generally observed between plasmid content and cognate spacer sequences, supporting the role of the CRISPR/Cas system in protecting against foreign DNA elements. However, in several group III strains, retention of plasmid pEU30 is inconsistent with a functional CRISPR/Cas system.
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Zhang, Xinfu, Sandra Garrett, Brenton R. Graveley und Michael P. Terns. „Unique properties of spacer acquisition by the type III-A CRISPR-Cas system“. Nucleic Acids Research 50, Nr. 3 (10.12.2021): 1562–82. http://dx.doi.org/10.1093/nar/gkab1193.
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Abstract Type III CRISPR-Cas systems have a unique mode of interference, involving crRNA-guided recognition of nascent RNA and leading to DNA and RNA degradation. How type III systems acquire new CRISPR spacers is currently not well understood. Here, we characterize CRISPR spacer uptake by a type III-A system within its native host, Streptococcus thermophilus. Adaptation by the type II-A system in the same host provided a basis for comparison. Cas1 and Cas2 proteins were critical for type III adaptation but deletion of genes responsible for crRNA biogenesis or interference did not detectably change spacer uptake patterns, except those related to host counter-selection. Unlike the type II-A system, type III spacers are acquired in a PAM- and orientation-independent manner. Interestingly, certain regions of plasmids and the host genome were particularly well-sampled during type III-A, but not type II-A, spacer uptake. These regions included the single-stranded origins of rolling-circle replicating plasmids, rRNA and tRNA encoding gene clusters, promoter regions of expressed genes and 5′ UTR regions involved in transcription attenuation. These features share the potential to form DNA secondary structures, suggesting a preferred substrate for type III adaptation. Lastly, the type III-A system adapted to and protected host cells from lytic phage infection.
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Kim, Jenny G., Sandra Garrett, Yunzhou Wei, Brenton R. Graveley und Michael P. Terns. „CRISPR DNA elements controlling site-specific spacer integration and proper repeat length by a Type II CRISPR–Cas system“. Nucleic Acids Research 47, Nr. 16 (08.08.2019): 8632–48. http://dx.doi.org/10.1093/nar/gkz677.
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Abstract 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.