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

Courtney M Hill; Asma Hatoum-Aslan

doi:10.1007/978-1-0716-3523-0_18

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

Methods Mol Biol. 2024:2734:279-299. doi: 10.1007/978-1-0716-3523-0_18.

Authors

Courtney M Hill¹, Asma Hatoum-Aslan²

Affiliations

¹ Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, IL, USA.
² Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, IL, USA. ahatoum@illinois.edu.

PMID: 38066376
DOI: 10.1007/978-1-0716-3523-0_18

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.

Keywords: Bacteriophage engineering; CRISPR-Cas10; Phage genome editing; Staphylococci; Type III-A CRISPR-Cas.

MeSH terms

Bacteriophages* / genetics
CRISPR-Cas Systems* / genetics
Genetic Engineering / methods