The utilization of bacteriophages - viruses that infect bacteria - presents an innovation frontier brimming with potential within the realms of biotechnology and medicine. Phages are the most abundant evolving systems on Earth, and as such, present a formidable reservoir of bioactive materials, including, among many other biological marvels, enzymes, and nanostructures that can be further tuned by laboratory engineering and evolution. Discovering, understanding, and harnessing resources from phages, has gathered momentum in the past two decades and led to groundbreaking discoveries. Yet, we still fall short of fully mastering phage engineering to rapidly introduce genomic variation at will, to assemble and to select phages independently of a living host, thus preventing tapping into phages’ full potential. Engineering the genome of lytic bacteriophages using in vivo methods, has long been a challenge due to the rapid cell death caused by phage infection, hindering efficient genetic manipulation. To circumvent this limitation, methods based on homologous recombination in yeast have been developed, albeit with cumbersome cloning steps. Lately, CRISPR methods have shown promise but have some limitations in downstream selection. As an alternative, in vitro cloning methods have gained traction by offering rapidity and precise control over DNA assembly. Despite technical barriers, such as genome cleanup, advances in DNA synthesis and sequencing have fostered the utilization of such methods. Transforming large DNA assemblies into desired hosts, however, remains difficult.
Addressing these challenges, Shingo Nozaki introduced a method utilizing lambda phage's packaging to encapsulate DNA assemblies [1], eliminating the need for transformation. This elegant solution, while limited to lambda-sized DNA assemblies, exemplifies a novel approach to phage engineering. Inspired by Nozaki and building upon our previous work in E. coli cell-free transcription-translation (TXTL), we developed PHEIGES (PHage Engineering by In vitro Gene Expression and Selection), a method enabling the seamless direct assembly of phage genomes from PCR-amplified fragments, subsequently expressed to produce engineered phages within a single day with very high yields (up to 10¹¹ PFU/ml), without purification steps [2].
PHEIGES integrates the advantages of yeast assembly speed, in vitro cloning precision, and CRISPR efficacy. Notably, we successfully re-assembled the genome of and synthesized the 86 kbp FelixO1 Salmonella phage, to date the largest rebooted phage in TXTL from its re-assembled genome. Furthermore, using the T7 phage model system, we showcased the simplicity of using PHEIGES for gene deletion, addition, and for introducing large mutation libraries to key phage functional proteins as the tail fiber, responsible for interacting with the bacterial host cell. PHEIGES was also devised to be affordable and thus accessible to many laboratories.
One of the compelling aspects of PHEIGES is its potential capacity to bridge the genotype-phenotype linkage of phage assembly in bulk transcription-translation systems. The cell-free synthesis of phages from engineered genomes exhibits a higher-than-random phenotype-genotype coupling, enabling the direct selection of host range variants from the tail fiber T7 assembled libraries without in vivo steps or any sort of compartmentalization. This observation was unexpected as phages are synthesized from assembled genome mix in batch mode TXTL and counters decades of practice when manipulating phage (display) libraries. It is tempting to hypothesize that phages may have an inherent capacity to self-assemble individually. This observation could be a result of (i) diffusion constraints of a fast-oligomerization system in TXTL reactions where the volume per phage is two orders of magnitude greater than within a cell and/or (ii) an evolutionary phage trait driven by minimizing protein synthesis to the benefit of maximizing phage production from limited cellular resources. A secondary selective advantage may be to accomplish genotype-phenotype coupling within a cell, where individual phage mutants may arise due to replication errors. This observation could not have been discovered in an in vivo system and serves as a starting point to probe both hypotheses. We anticipate that the observed imperfect phenotype-genotype coupling can be further improved by optimizing the biochemistry and biophysics of TXTL reaction conditions coupled with lab evolution.
Currently, PHEIGES applies to phages that can be successfully synthesized in TXTL and enables rebooting engineered phages with genomes up to 86 kbp. The development of TXTL systems from microorganisms other than E. coli could expand the current spectrum of PHEIGES. Among other limitations to be addressed are the presence of DNA modifications in some phage genomes (e.g., T4).
By enabling precise, rapid, and scalable engineering of bacteriophages, this method opens new avenues for the exploration and exploitation of these viral entities. PHEIGES holds promise for the engineering of a broader range of bacteriophages, including those targeting non-E. coli hosts and for tailoring phages to act on specific hosts, as pathogens (e.g., phage therapy). It opens avenues for radical phage hybridization and for probing large DNA assemblies in TXTL to decipher new phage defense systems like anti-CRISPR. Moreover, this work underscores the potential of TXTL for reconstituting complex gene circuits, minimal genomes for synthetic cells, and protein machinery in vitro, transcending phage research to broader genetic engineering applications. PHEIGES offers a versatile and efficient platform for exploring the intricate landscape of phage genomes and beyond. As demonstrated with phage T7, PHEIGES also enables revealing the activity of enzymes encoded into genome parts only. Pairing PHEIGES with bioinformatics tools could accelerate the discovery of new phage enzymes with a broad application spectrum.
[1] Nozaki, S. Rapid and Accurate Assembly of Large DNA Assisted by In Vitro Packaging of Bacteriophage. ACS Synth Biol 11, 4113–4122 (2022).
[2] Levrier, A., Karpathakis, I., Nash, B., Bowden, S.D., Lindner, A.B., Noireaux, V. PHEIGES: All Cell-Free Phage Synthesis and Selection from Engineered Genomes. Nat Comm 15(1):2223 (2024).