Abstract
In recent years, bacteriophage research has been boosted by a rising interest in using phage therapy to treat antibiotic-resistant bacterial infections. In addition, there is a desire to use phages and their unique proteins for specific biocontrol applications and diagnostics. However, the ability to manipulate phage genomes to understand and control gene functions, or alter phage properties such as host range, has remained challenging due to a lack of universal selectable markers. Here, we discuss the state-of-the-art techniques to engineer and select desired phage genomes using advances in cell-free methodologies and clustered regularly interspaced short palindromic repeats-CRISPR associated protein (CRISPR-Cas) counter-selection approaches.
Clustered regularly interspaced short palindromic repeats-CRISPR associated protein (CRISPR-Cas) systems are efficient at counter-selecting mutant phages generated by homologous recombination-based approaches, but are still limited by spacer efficiency and CRISPR-evading strategies of phages.RNA-targeting CRISPR-Cas systems, such as type III and VI, can provide a more robust selection tool, especially for phages that evolved strategies to protect their DNA from targeting. Furthermore, these systems avoid the enrichment of CRISPR escape mutants.Ex vivo phage genome engineering strategies assemble and reboot synthetic phage genomes and allow for increased flexibility in the design of the phage genome.Rebooting of synthetic phage genomes is facilitated in cell-free systems by the exclusion of the cell membrane barrier, but it is currently restricted to a very small number of bacterial species.