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

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Títol: CRISPR-spacer integration reporter plasmids reveal distinct genuine acquisition specificities among CRISPR-Cas I-E variants of Escherichia coli
Autors: Díez-Villaseñor, César | Guzmán, Noemí M. | Almendros, Cristóbal | García-Martínez, Jesús | Mojica, Francisco J.M.
Grups d'investigació o GITE: Microbiología Molecular
Centre, Departament o Servei: Universidad de Alicante. Departamento de Fisiología, Genética y Microbiología
Paraules clau: Protospacer adjacent motif | Cas genes | Cascade | CRISPR-spacer acquisition | Reporter plasmids | RNA-guided immunity | Spacer orientation | Ruler mechanism | Escherichia coli K12 | O157:H7
Àrees de coneixement: Microbiología
Data de publicació: de maig-2013
Editor: Landes Bioscience
Citació bibliogràfica: RNA Biology 2013; 10:792-802; PMID: 23445770; http://dx.doi.org/10.4161/rna.24023
Resum: Prokaryotes 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 mechanism.
Patrocinadors: This work was funded by the Ministerio de Economía y Competitividad (BIO2011-24417).
URI: http://hdl.handle.net/10045/36087
ISSN: 1547-6286 (Print) | 1555-8584 (Online)
DOI: 10.4161/rna.24023
Idioma: eng
Tipus: info:eu-repo/semantics/article
Drets: This 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 cited.
Revisió científica: si
Versió de l'editor: http://dx.doi.org/10.4161/rna.24023
Apareix a la col·lecció: INV - Microbiología Molecular - Artículos de Revistas

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