Synthetic and Functional Engineering of Bacteriophages: Approaches for Tailored Bactericidal, Diagnostic, and Delivery Platforms

Abstract

Bacteriophages (phages), the most abundant biological entities on Earth, have long served as both model systems and therapeutic tools. Recent advances in synthetic biology and genetic engineering have revolutionized the capacity to tailor phages with enhanced functionality beyond their natural capabilities. This review outlines the current landscape of synthetic and functional engineering of phages, encompassing both in-vivo and in-vitro strategies. We describe in-vivo approaches such as phage recombineering systems, CRISPR-Cas-assisted editing, and bacterial retron-based methods, as well as synthetic assembly platforms including yeast-based artificial chromosomes, Gibson, Golden Gate, and iPac assemblies. In addition, we explore in-vitro rebooting using TXTL (transcription-translation) systems, which offer a flexible alternative to cell-based rebooting but are less effective for large genomes or structurally complex phages. Special focus is given to the design of customized phages for targeted applications, including host range expansion via receptor-binding protein modifications, delivery of antimicrobial proteins or CRISPR payloads, and the construction of biocontained, non-replicative capsid systems for safe clinical use. Through illustrative examples, we highlight how these technologies enable the transformation of phages into programmable bactericidal agents, precision diagnostic tools, and drug delivery vehicles. Together, these advances establish a powerful foundation for next-generation antimicrobial platforms and synthetic microbiology.

Keywords: CRISPR-Cas systems; antimicrobial delivery platforms; bacteriophage engineering; cell-free TXTL systems; host range expansion; non-replicative phage; phage assembly and rebooting; phage recombineering; retron-mediated editing; synthetic biology.

Figure 1

Graphical Abstract of Phage Engineering Strategies and Representative Applications. (A) Overview of available phage engineering approaches categorized into in-vivo and in-vitro methods. (B) Representative applications of engineered phages, including (1) expanding host range via modification of phage tail fiber proteins to express alternative receptor binding proteins (RBPs); (2) bacterial detection through the incorporation of reporter genes or molecular tags; (3) delivery of CRISPR-Cas systems for targeted bacterial elimination or genome editing; and (4) in situ expression of therapeutic proteins for antimicrobial or modulatory purposes.

Figure 2

In-vivo Methods for Phage Engineering with Representative Mechanisms. (A) Non-assisted phage engineering through spontaneous homologous recombination between phage and donor DNA without auxiliary genetic tools. (B) Assisted phage engineering strategies, classified by the supporting system employed: (B1) the λ red recombineering system, utilizing Exo, Beta, and Gam proteins to facilitate homologous recombination with linear or circular donor DNA (ssDNA or dsDNA). (B2) CRISPR-Cas-assisted engineering system, where targeted double-stranded DNA breaks (DSBs) enhance recombination efficiency and eliminate wild-type (WT) phage via counterselection. (B3) Retron-based engineering, where bacterial retrons generate donor ssDNA through reverse transcription of ncRNA comprising msr and msd regions. In all panels, donor DNA is depicted in blue, homologous regions (HRs) in red, Cas nucleases in purple, and guide RNAs in light red. Phage genetic material may be introduced as infectious particles or via electroporation. These platforms enable precise genome editing in both temperate and lytic phages.

Figure 3

Overview of Synthetic Phage Engineering Strategies: Assembly and Rebooting Options. This figure shows the two main steps in synthetic phage engineering: genome assembly and phage rebooting. (A) YAC-based assembly performed in yeast cells (in-vivo). (B) Gibson assembly performed in-vitro using DNA fragments with overlapping ends. (C) In-vivo rebooting by transforming assembled genomes into native hosts, intermediate strains, or L-form bacteria. (D) In-vitro rebooting using TXTL (cell-free transcription–translation) systems. Steps (A,C) are in-vivo methods; (B,D) are in-vitro methods. Resulting phages can be further propagated in suitable bacterial hosts.

Figure 4

Recent Advancements in Antibacterial Phage Capsid Systems. (A) CRISPR-Cas12a-based antimicrobial capsid system using the temperate λ phage [24]. (B) Biocontained antibacterial capsid system using the lytic T7 phage to deliver colicin E1 [41]. In (A), a CRISPR-Cas12a plasmid lacking a primase and antibiotic resistance gene. The plasmid includes a phage origin of replication and a thymidylate synthase gene (thyA) for selection in a ΔthyA host, and it is packaged into λ phage capsids via the cos packaging signal. Tail-related genes are STF and gpJ, which encode the λ side tail fiber and tail tip proteins, respectively. In (B), a BAC-based helper plasmid expresses the T7 phage structural genes. The remaining T7 genome, including replication and packaging genes along with the colicin E1 expression cassette, is assembled separately and introduced into a bacterial strain carrying the helper BAC plasmid for particle production.

Figure 5

Chronological Milestones in The Development of Phage Engineering Systems from The Early 20th Century to Future Prospects. The lower section of the figure shows selected key discoveries and milestones that contributed to the current advancement of phage engineering, while the upper section describes the current and future expected advancement of synthetic phage engineering for therapeutic applications. The upper section begins with the discovery of phages [3,4] and it lists key achievements and innovations such as: the confirmation of phage crossing between closely related phages [11], the discovery of the λ red system [85], the complete sequencing of the first phage [15], the invention of phage display [190], and the Yeast Artificial Chromosome system (YAC) [154]. It also includes the first application of the λ red system in phage engineering [94], the innovations of Golden Gate assembly [149] and Gibson assembly [148], the assembling of phage Φx174 in-vitro [191], the assembling of Φx174 in-vivo using YAC system [147], and the development of E. coli TX-TL platforms for phage rebooting [152,192]. Additionally, it covers the rebooting of T4 phage using the TX-TL method and concludes with the global burst in whole-genome sequencing and the availability of large genomic data, which is leading to a new era in phage engineering. As machine learning and artificial intelligence are integrated into phage engineering systems, we anticipate reaching a new era of phage engineering platforms [180].