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      CRISPRStudio: A User-Friendly Software for Rapid CRISPR Array Visualization

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          Abstract

          The CRISPR-Cas system biologically serves as an adaptive defense mechanism against phages. However, there is growing interest in exploiting the hypervariable nature of the CRISPR locus, often of viral origin, for microbial typing and tracking. Moreover, the spacer content of any given strain provides a phage resistance profile. Large-scale CRISPR typing studies require an efficient method for showcasing CRISPR array similarities across multiple isolates. Historically, CRISPR arrays found in microbes have been represented by colored shapes based on nucleotide sequence identity and, while this approach is now routinely used, only scarce computational resources are available to automate the process, making it very time-consuming for large datasets. To alleviate this tedious task, we introduce CRISPRStudio, a command-line tool developed to accelerate CRISPR analysis and standardize the preparation of CRISPR array figures. It first compares nucleotide spacer sequences present in a dataset and then clusters them based on sequence similarity to assign a meaningful representative color. CRISPRStudio offers versatility to suit different biological contexts by including options such as automatic sorting of CRISPR loci and highlighting of shared spacers, while remaining fast and user-friendly.

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          Most cited references35

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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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            Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product.

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              Interference by clustered regularly interspaced short palindromic repeat (CRISPR) RNA is governed by a seed sequence.

              Prokaryotic clustered regularly interspaced short palindromic repeat (CRISPR)/Cas (CRISPR-associated sequences) systems provide adaptive immunity against viruses when a spacer sequence of small CRISPR RNA (crRNA) matches a protospacer sequence in the viral genome. Viruses that escape CRISPR/Cas resistance carry point mutations in protospacers, though not all protospacer mutations lead to escape. Here, we show that in the case of Escherichia coli subtype CRISPR/Cas system, the requirements for crRNA matching are strict only for a seven-nucleotide seed region of a protospacer immediately following the essential protospacer-adjacent motif. Mutations in the seed region abolish CRISPR/Cas mediated immunity by reducing the binding affinity of the crRNA-guided Cascade complex to protospacer DNA. We propose that the crRNA seed sequence plays a role in the initial scanning of invader DNA for a match, before base pairing of the full-length spacer occurs, which may enhance the protospacer locating efficiency of the E. coli Cascade complex. In agreement with this proposal, single or multiple mutations within the protospacer but outside the seed region do not lead to escape. The relaxed specificity of the CRISPR/Cas system limits escape possibilities and allows a single crRNA to effectively target numerous related viruses.
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                Author and article information

                Journal
                Viruses
                Viruses
                viruses
                Viruses
                MDPI
                1999-4915
                01 November 2018
                November 2018
                : 10
                : 11
                : 602
                Affiliations
                [1 ]Département de Biochimie, de Microbiologie, et de Bio-informatique, Faculté des Sciences et de Génie, Université Laval, Québec City, QC G1V 0A6, Canada; moira.dion.1@ 123456ulaval.ca
                [2 ]Groupe de Recherche en Écologie Buccale, Faculté de Médecine Dentaire, Université Laval, Québec City, QC G1V 0A6, Canada
                [3 ]SyntBioLab Inc., 4820 rue de la Pascaline, Lévis, QC G6W 0L9, Canada; simon.labrie@ 123456syntbiolab.com
                [4 ]COPSAC, Copenhagen Prospective Studies on Asthma in Childhood, Herlev and Gentofte Hospital, University of Copenhagen, Ledreborg Alle 34, 2820 Gentofte, Denmark; shiraz.shah@ 123456dbac.dk
                Author notes
                [* ]Correspondence: sylvain.moineau@ 123456bcm.ulaval.ca ; Tel.: +1-418-656-3712
                Author information
                https://orcid.org/0000-0002-2832-5101
                Article
                viruses-10-00602
                10.3390/v10110602
                6267562
                30388811
                6d08a6bb-ef3d-42be-8b98-af08007a9a74
                © 2018 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                : 10 October 2018
                : 30 October 2018
                Categories
                Article

                Microbiology & Virology
                crispr,typing,software,visualization,viral origin,phage
                Microbiology & Virology
                crispr, typing, software, visualization, viral origin, phage

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