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Genome Editing in Staphylococcus aureus by Conditional Recombineering and CRISPR/Cas9-Mediated Counterselection

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Electroporation Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2050))

Abstract

Methods for the genetic manipulation of S. aureus have historically proven challenging, which has hindered experimental studies of this organism. We recently developed a system for recombineering and CRISPR/Cas9-mediated counterselection in S. aureus which utilizes commercially synthesized synthetic DNA oligonucleotides as substrates for introducing precise genomic modifications into the organism and for performing lethal counterselection of unedited cells. These techniques make it possible to scalably and inexpensively engineer desired genomic changes into laboratory or clinical S. aureus strains, using electroporation to introduce the effector plasmid vectors and oligonucleotides. Here we describe detailed protocols for performing genome editing of S. aureus in order to produce isogenic strains using this system and detail general principles which are broadly applicable across a range of organisms for which equivalent systems have been established.

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Author information

Authors and Affiliations

  1. Department of Laboratory Medicine, University of Washington, Seattle, WA, USA

    Kelsi Penewit & Stephen J. Salipante

Authors
  1. Kelsi Penewit
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  2. Stephen J. Salipante
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Corresponding author

Correspondence to Stephen J. Salipante .

Editor information

Editors and Affiliations

  1. MD Anderson Cancer Center, The University of Texas, Houston, TX, USA

    Shulin Li

  2. School of Biological Science and Medical Engineering, Beihang University, Beijing, China, Institute of Nanotechnology for Single Cell Analysis (INSCA), Beijing Advanced Innovation Center for Biomedical Engineering, Beihang University,, Beijing, China

    Lingqian Chang

  3. Institut de Pharmacologie et de Biologie Structurale, Université de Toulouse, CNRS, UPS, Toulouse, France

    Justin Teissie

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Penewit, K., Salipante, S.J. (2020). Genome Editing in Staphylococcus aureus by Conditional Recombineering and CRISPR/Cas9-Mediated Counterselection. In: Li, S., Chang, L., Teissie, J. (eds) Electroporation Protocols. Methods in Molecular Biology, vol 2050. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9740-4_14

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  • DOI: https://doi.org/10.1007/978-1-4939-9740-4_14

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9739-8

  • Online ISBN: 978-1-4939-9740-4

  • eBook Packages: Springer Protocols

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