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Genetic Engineering of Therapeutic Phages Using Type III CRISPR-Cas Systems

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Bacteriophage Therapy

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2734))

Abstract

The functional characterization of “hypothetical” phage genes is a major bottleneck in basic and applied phage research. To compound this issue, the most suitable phages for therapeutic applications—the strictly lytic variety—are largely recalcitrant to classical genetic techniques due to low recombination rates and lack of selectable markers. Here we describe methods for fast and effective phage engineering that rely upon a Type III-A CRISPR-Cas system. In these methods, the CRISPR-Cas system is used as a powerful counterselection tool to isolate rare phage recombinants.

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Acknowledgments

A. H.-A. holds an Investigators in the Pathogenesis of Infectious Disease Award from the Burroughs Wellcome Fund. She is also supported by an NSF/MCB CAREER award [2054755] and NIH/NIAID [R21AI156636-01].

Declaration of Interests

A. H.-A. is the inventor of a US patent relating to this work (US20180251787A1).

Author information

Authors and Affiliations

  1. Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, IL, USA

    Courtney M. Hill & Asma Hatoum-Aslan

Authors
  1. Courtney M. Hill
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  2. Asma Hatoum-Aslan
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Corresponding author

Correspondence to Asma Hatoum-Aslan .

Editor information

Editors and Affiliations

  1. Centre of Biological Engineering, University of Minho, Braga, Portugal

    Joana Azeredo

  2. International Iberian Nanotechnology Laboratory, Braga, Portugal

    Sanna Sillankorva

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© 2024 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Hill, C.M., Hatoum-Aslan, A. (2024). Genetic Engineering of Therapeutic Phages Using Type III CRISPR-Cas Systems. In: Azeredo, J., Sillankorva, S. (eds) Bacteriophage Therapy. Methods in Molecular Biology, vol 2734. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3523-0_18

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  • DOI: https://doi.org/10.1007/978-1-0716-3523-0_18

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3522-3

  • Online ISBN: 978-1-0716-3523-0

  • eBook Packages: Springer Protocols

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