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Design and Construction of a Recombinant pTargetF Vector Carrying an entB gRNA for the CRISPR/Cas9 in E. coli Serogroup O2
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Mohadese Amiri    , Mehdi Golchin * , Abbas Hajizade , Hamidreza Farzin , Majid Jamshidian-Mojaver |
| Department of Pathobiology, Faculty of Veterinary Medicine, Shahid Bahonar University of Kerman, Kerman, Iran. , golchin@uk.ac.ir |
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Abstract: (302 Views) |
Colibacillosis caused by Escherichia coli serotype O2 is one of the major bacterial diseases in the poultry industry, leading to substantial economic losses. The increasing prevalence of antibiotic resistance among pathogenic strains has highlighted the necessity for alternative strategies, including the development of vaccines and attenuated strains. The aim of this study was to design and construct a recombinant CRISPR/Cas9 vector carrying a specific gRNA targeting the entB gene in E. coli serotype O2, as an initial step toward the development of an attenuated strain. In this study, a specific gRNA against the entB gene was designed using the CHOPCHOP web tool. The forward and reverse oligonucleotides corresponding to the gRNA were synthesized and, following annealing, were ligated into the BbsI-linearized pTargetF plasmid using T4 DNA ligase. The resulting recombinant vector was then transformed into competent DH5α cells. The successful insertion of the gRNA fragment into the vector was confirmed by polymerase chain reaction (PCR) and subsequent sequencing. The results demonstrated that the designed guide sequence was successfully cloned into the pTargetF vector. The observation of the expected bands in PCR analysis confirmed the correct construction of the recombinant construct. Based on the findings of this study, the designed recombinant vector can serve as a preliminary tool for targeting the entB gene and for future investigations aimed at attenuating pathogenic E. coli strains in poultry.
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| Keywords: Escherichia coli, serogroup O2, CRISPR/Cas9, entB, gRNA, recombinant vector, colibacillosis |
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Full-Text [PDF 1112 kb]
(37 Downloads)
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Type of Study: Research |
Subject:
Microrganisms and Viruses
Received: 2026/02/20 | Accepted: 2026/03/19 | Published: 2026/03/19
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Amiri M, Golchin M, Hajizade A, Farzin H, Jamshidian-Mojaver M. Design and Construction of a Recombinant pTargetF Vector Carrying an entB gRNA for the CRISPR/Cas9 in E. coli Serogroup O2. gebsj 2026; 14 (2) :269-281 URL: http://gebsj.ir/article-1-554-en.html
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