CRISPR-spacer integration reporter plasmids reveal distinct genuine acquisition specificities among CRISPR-Cas I-E variants of Escherichia coli

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dc.contributorMicrobiología Moleculares
dc.contributor.authorDíez-Villaseñor, César-
dc.contributor.authorGuzmán, Noemí M.-
dc.contributor.authorAlmendros, Cristóbal-
dc.contributor.authorGarcía-Martínez, Jesús-
dc.contributor.authorMojica, Francisco J.M.-
dc.contributor.otherUniversidad de Alicante. Departamento de Fisiología, Genética y Microbiologíaes
dc.identifier.citationRNA Biology 2013; 10:792-802; PMID: 23445770;
dc.identifier.issn1547-6286 (Print)-
dc.identifier.issn1555-8584 (Online)-
dc.description.abstractProkaryotes immunize themselves against transmissible genetic elements by the integration (acquisition) in clustered regularly interspaced short palindromic repeats (CRISPR) loci of spacers homologous to invader nucleic acids, defined as protospacers. Following acquisition, mono-spacer CRISPR RNAs (termed crRNAs) guide CRISPR-associated (Cas) proteins to degrade (interference) protospacers flanked by an adjacent motif in extrachomosomal DNA. During acquisition, selection of spacer-precursors adjoining the protospacer motif and proper orientation of the integrated fragment with respect to the leader (sequence leading transcription of the flanking CRISPR array) grant efficient interference by at least some CRISPR-Cas systems. This adaptive stage of the CRISPR action is poorly characterized, mainly due to the lack of appropriate genetic strategies to address its study and, at least in Escherichia coli, the need of Cas overproduction for insertion detection. In this work, we describe the development and application in Escherichia coli strains of an interference-independent assay based on engineered selectable CRISPR-spacer integration reporter plasmids. By using this tool without the constraint of interference or cas overexpression, we confirmed fundamental aspects of this process such as the critical requirement of Cas1 and Cas2 and the identity of the CTT protospacer motif for the E. coli K12 system. In addition, we defined the CWT motif for a non-K12 CRISPR-Cas variant, and obtained data supporting the implication of the leader in spacer orientation, the preferred acquisition from plasmids harboring cas genes and the occurrence of a sequential cleavage at the insertion site by a ruler
dc.description.sponsorshipThis work was funded by the Ministerio de Economía y Competitividad (BIO2011-24417).es
dc.publisherLandes Biosciencees
dc.rightsThis is an open-access article licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported License. The article may be redistributed, reproduced, and reused for non-commercial purposes, provided the original source is properly
dc.subjectProtospacer adjacent motifes
dc.subjectCas geneses
dc.subjectCRISPR-spacer acquisitiones
dc.subjectReporter plasmidses
dc.subjectRNA-guided immunityes
dc.subjectSpacer orientationes
dc.subjectRuler mechanismes
dc.subjectEscherichia coli K12es
dc.titleCRISPR-spacer integration reporter plasmids reveal distinct genuine acquisition specificities among CRISPR-Cas I-E variants of Escherichia colies
Appears in Collections:INV - Microbiología Molecular - Artículos de Revistas

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