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      An updated evolutionary classification of CRISPR–Cas systems

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          Abstract

          The evolution of CRISPR– cas loci, which encode adaptive immune systems in archaea and bacteria, involves rapid changes, in particular numerous rearrangements of the locus architecture and horizontal transfer of complete loci or individual modules. These dynamics complicate straightforward phylogenetic classification, but here we present an approach combining the analysis of signature protein families and features of the architecture of cas loci that unambiguously partitions most CRISPR– cas loci into distinct classes, types and subtypes. The new classification retains the overall structure of the previous version but is expanded to now encompass two classes, five types and 16 subtypes. The relative stability of the classification suggests that the most prevalent variants of CRISPR–Cas systems are already known. However, the existence of rare, currently unclassifiable variants implies that additional types and subtypes remain to be characterized.

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          Most cited references84

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          CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA.

          Horizontal gene transfer (HGT) in bacteria and archaea occurs through phage transduction, transformation, or conjugation, and the latter is particularly important for the spread of antibiotic resistance. Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci confer sequence-directed immunity against phages. A clinical isolate of Staphylococcus epidermidis harbors a CRISPR spacer that matches the nickase gene present in nearly all staphylococcal conjugative plasmids. Here we show that CRISPR interference prevents conjugation and plasmid transformation in S. epidermidis. Insertion of a self-splicing intron into nickase blocks interference despite the reconstitution of the target sequence in the spliced mRNA, which indicates that the interference machinery targets DNA directly. We conclude that CRISPR loci counteract multiple routes of HGT and can limit the spread of antibiotic resistance in pathogenic bacteria.
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            Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin.

            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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              Phage response to CRISPR-encoded resistance in Streptococcus thermophilus.

              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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                Author and article information

                Journal
                101190261
                31733
                Nat Rev Microbiol
                Nat. Rev. Microbiol.
                Nature reviews. Microbiology
                1740-1526
                1740-1534
                20 March 2017
                28 September 2015
                November 2015
                11 May 2017
                : 13
                : 11
                : 722-736
                Affiliations
                [1 ]National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Maryland 20894, USA
                [2 ]Bioinformatics group, Department of Computer Science, University of Freiberg, Georges-Kohler-Allee 106, 79110 Freiberg, Germany
                [3 ]Archaea Centre, Department of Biology, Copenhagen University, Ole Maaløes Vej 5, DK2200 Copenhagen N, Denmark
                [4 ]Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Raleigh, North Carolina 27606, USA
                [5 ]Laboratory of Microbiology, Wageningen University, Dreijenplein 10, 6703HB Wageningen, Netherlands
                [6 ]Department of Regulation in Infection Biology, Helmholtz Centre for Infection Research, D-38124 Braunschweig, Germany
                [7 ]DuPont Nutrition and Health, BP10, Dangé-Saint-Romain 86220, France
                [8 ]Département de Biochimie, de Microbiologie et de Bio-informatique, Faculté des Sciences et de Génie, Groupe de Recherche en Écologie Buccale, Félix d’Hérelle Reference Center for Bacterial Viruses, Faculté de médecine dentaire, Université Laval, Québec City, Québec, Canada
                [9 ]Departamento de Fisiología, Genética y Microbiología. Universidad de Alicante. 03080-Alicante, Spain
                [10 ]Biochemistry and Molecular Biology, Genetics and Microbiology, University of Georgia, Davison Life Sciences Complex, Green Street, Athens, Georgia 30602, USA
                [11 ]Biomedical Sciences Research Complex, University of St Andrews, North Haugh, St Andrews, KY16 9TZ, UK
                [12 ]Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, M5S 3E5, Canada
                [13 ]BIOSS Centre for Biological Signaling Studies, Cluster of Excellence, University of Freiburg, Germany
                Author notes
                Correspondence to E.V.K. koonin@ 123456ncbi.nlm.nih.gov
                Article
                PMC5426118 PMC5426118 5426118 nihpa855430
                10.1038/nrmicro3569
                5426118
                26411297
                750f59c6-1a9d-4fa3-9506-1f5e5d0c7a2d
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