Background: The therapeutic potential of bacteriophages has been debated since their first isolation and characterisation in the early 20 th century. However, a lack of consistency in application and observed efficacy during their early use meant that upon the discovery of antibiotic compounds research in the field of phage therapy quickly slowed. The rise of antibiotic resistance in bacteria and improvements in our abilities to modify and manipulate DNA, especially in the context of small viral genomes, has led to a recent resurgence of interest in utilising phage as antimicrobial therapeutics.Results: In this article a number of results from the literature that have aimed to address key issues regarding the utility and efficacy of phage as antimicrobial therapeutics utilising molecular biology and synthetic biology approaches will be introduced and discussed, giving a general view of the recent progress in the field.Conclusions: Advances in molecular biology and synthetic biology have enabled rapid progress in the field of phage engineering, with this article highlighting a number of promising strategies developed to optimise phages for the treatment of bacterial disease. Whilst many of the same issues that have historically limited the use of phages as therapeutics still exist, these modifications, or combinations thereof, may form a basis upon which future advances can be built. A focus on rigorous in vivo testing and investment in clinical trials for promising candidate phages may be required for the field to truly mature, but there is renewed hope that the potential benefits of phage therapy may finally be realised.

The discovery of clustered, regularly interspaced, short palindromic repeats (CRISPR) and the Cas9 RNA-guided nuclease provides unprecedented opportunities to selectively kill specific populations or species of bacteria. However, the use of CRISPR-Cas9 to clear bacterial infections in vivo is hampered by the inefficient delivery of cas9 genetic constructs into bacterial cells. Here, we use a broad-host-range P1-derived phagemid to deliver the CRISPR-Cas9 chromosomal-targeting system into Escherichia coli and the dysentery-causing Shigella flexneri to achieve DNA sequence-specific killing of targeted bacterial cells. We show that genetic modification of the helper P1 phage DNA packaging site (pac) significantly enhances the purity of packaged phagemid and improves the Cas9-mediated killing of S. flexneri cells. We further demonstrate that P1 phage particles can deliver chromosomaltargeting cas9 phagemids into S. flexneri in vivo using a zebrafish larvae infection model, where they significantly reduce the bacterial load and promote host survival. Our study highlights the potential of combining P1 bacteriophage-based delivery with the CRISPR chromosomal-targeting system to achieve DNA sequence-specific cell lethality and efficient clearance of bacterial infection.

The discovery of clustered, regularly interspaced, short palindromic repeats (CRISPR) and the Cas9 RNA-guided nuclease provides unprecedented opportunities to selectively kill specific populations or species of bacteria. However, the use of CRISPR-Cas9 to clear bacterial infections in vivo is hampered by the inefficient delivery of cas9 genetic constructs into bacterial cells. Here, we use a broad-host-range P1-derived phagemid to deliver the CRISPR-Cas9 chromosomal-targeting system into Escherichia coli and the dysentery-causing Shigella flexneri to achieve DNA sequence-specific killing of targeted bacterial cells. We show that genetic modification of the helper P1 phage DNA packaging site (pac) significantly enhances the purity of packaged phagemid and improves the Cas9-mediated killing of S. flexneri cells. We further demonstrate that P1 phage particles can deliver chromosomal-targeting cas9 phagemids into S. flexneri in vivo using a zebrafish larvae infection model, where it significantly reduces the bacterial load and promotes host survival. Our study highlights the potential of combining a P1 bacteriophage-based delivery with the CRISPR chromosomal-targeting system to achieve DNA sequence-specific cell lethality and efficient clearance of bacterial infection.

The lytic replication of bacteriophage P1 requires RepL expression and the lytic stage origin, oriL, which is postulated to be located within repL gene sequence. The exact sequence of P1 oriL and the mechanism(s) of RepL-mediated DNA replication, however, are not fully understood. By using repL gene expression to induce DNA replication of a gfp and a rfp reporter plasmids, we demonstrated that synonymous base substitution in an adenine/thymidine-rich region of repL gene sequence, termed AT2, significantly inhibited the RepL-mediated signal amplification. Contrastingly, mutations in an IHF and two DnaA binding sites did not affect the RepL-mediated signal amplification significantly. A truncated repL sequence with the AT2 region allowed RepL-mediated signal amplification in trans therefore verifying a significant role of the AT2 region in RepL-mediated DNA replication. A combination of repL gene expression and a non-protein-coding copy of repL gene sequence (termed nc-repL) was able to amplify the output of an arsenic biosensor. Furthermore, mutation(s) at single or multiple positions within the AT2 region produced varying levels of RepL-mediated signal amplification. Overall, our results provide novel insights into the identity and location of P1 oriL as well as demonstrating the potential of using repL constructs to amplify and modulate the output of genetic biosensors.