Approaches for bacteriophage genome engineering

A novel T4- and λ-based receptor binding protein family for bacteriophage therapy host range engineering

“…It is worth noting, however, that while initial absorption to a bacterial surface often serves as the greatest filter to host range specificity there are a suite of other considerations in the engineering of successful recombinant phage. For example, proper DNA injection and transfer, native bacterial anti-viral defenses (e.g., CRISPR-Cas9 or Toxin-Antitoxin altruistic suicide cassettes) or proper development of phage can all be rate-limiting steps in bacteriophage infection and propagation post-absorption ( Pires et al, 2016 ; Łobocka et al, 2021 ).Various phage engineering techniques have been pioneered to address these issues including a wide range of phage genome engineering strategies to circumvent native antibacterial resistances or non-compatible genomic islands or GC contents for more efficient phage co-opting of bacterial host replication machinery ( Duong et al, 2020 ; Mahler et al, 2022 ). This having been said, recombinant expression of the modular RBP and tail fiber assembly lineage reported here might serve to help expand the host range of several very well characterized phages to several clinically important human pathogens for use in therapeutic applications and be of notable interest for phage-based technologies at large.…”

Bacteriophages as nanocarriers for targeted drug delivery and enhanced therapeutic effects

“…28 Additionally, phage genome modification involving advanced technologies employs manipulating the genetic material of bacteriophages to enhance the lytic profile. 98 Studies on the surface engineering of bacteriophages for drug delivery are described below and summarised in Table 2.…”

Continuous Multiplexed Phage Genome Editing Using Recombitrons

“…Propagation of phages through such a complex culture leads to the accumulation of multiple distinct edits at distant locations in individual phage genomes. Here, we demonstrate this approach, showing that (1) the editing is a continuous process in which edits accumulate over time; (2) it can be applied to multiple types of phage and used to introduce different edit types; (3) it can be optimized to reach efficiencies that do not require counterselection; and (4) it can be used to make multiplexed edits across a phage genome. For disambiguation with other techniques, we call this approach phage retron recombineering, and term the molecular components that include a modified retron a recombitron.…”

“…The various approaches to modify phage genomes and the limitations imposed by each have been recently reviewed 2 . One approach is to modify phage genomes by recombination within their bacterial host, which is inefficient and requires laborious screening of phage plaques.…”

“…Phage genome editing is a critical tool to engineer more effective phage technologies. However, editing phage genomes has traditionally been a low efficiency process that requires laborious screening, counter selection, or in vitro construction of modified genomes 2 . These requirements impose limitations on the type and throughput of phage modifications, which in turn limit our knowledge and potential for innovation.…”