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Poster De Conférence Année : 2023

Engineering Staphylococcus aureus phage K genome using CRISPR-Cas9

Résumé

The increasing prevalence of multidrug resistant pathogens within the microbial world, in addition to the high tolerance of biofilms to antibiotics and common disinfectants, makes it urgent to develop new treatment methods for antibiotic-resistant bacteria and biofilms. Among the different alternatives to antibiotics currently being explored, phage therapy appears to be one of the most promising. The limitations inherent to phages, such as their host range restriction or their moderate antimicrobial and antibiofilm efficacy can be overcome thanks to recent advances in genetic engineering and synthetic biology. In this work, we intend to develop a CRISPR-Cas9 assisted engineering approach to allow the development of engineered Staphylococcus aureus phages. First, five crRNA targeting the Major Capside Protein (MCP) of Staphylococcus aureus phage K were designed using Geneious and assembled using Golden Gate assembly into the pCasSA vector. Chemically competent Escherichia coli DH10B were transformed with the recombinant vectors and Midiprep of each recombinant plasmid were prepared. Correctly assembled pCasSA vector with their respective crRNA were screened and confirmed via BsaI restriction. Then, S. aureus RN4220 were transformed with each construct using an innovative non-electroporation transformation method. This method allowed the transformation of S. aureus bacteria with an efficiency of 7.5 102  1.5 102 UFC/108 viable cells. The efficiency of crRNA to cleave phage K genomes was determined by measuring the Efficiency Of Plating reduction (EOP). Among the five crRNA tested, three led to an EOP decrease comprised between two to three log. These first results demonstrate the possibility of implementing the CRISPR-Cas9 system inside S. aureus bacterial cells to target phage genomes. We plan to couple this system with homologous recombination approaches as a tool to create innovative diagnostic and therapeutic S. aureus engineered bacteriophages.
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Dates et versions

hal-04448695 , version 1 (09-02-2024)

Identifiants

  • HAL Id : hal-04448695 , version 1

Citer

Kandas Traore, Charles Julie, Carole Armal, Violeta Rodriguez-Ruiz, Damien Seyer, et al.. Engineering Staphylococcus aureus phage K genome using CRISPR-Cas9. Phages in Lyon 2023, Nov 2023, Lyon, France. ⟨hal-04448695⟩
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