Bacteriophages (phages) are a kind of bacterial viruses that usually lethal to their specific host bacteria. Phages are broadly used as effective biocontrol and sensitive diagnostics tools to lyse and detect the pathogens. Some phages, such as T-like and M13 phages, are common model organisms used in molecular biology research. (Figure1)
Figure 1A. Phage M13, which is used in phage display and gene engineering.
Figure 1B. Phage T4, which is a common lytic phage that have been deeply studied.
Phage therapy (PT) is considered as one of the most potential methodologies for fighting against multi-drug-resistant (MDR) bacteria clinically in the post-antibiotic era when classical antibiotic therapy is increasingly ineffective(Kmietowicz, 2017)(Gordillo Altamirano & Barr, 2019). Compared with the antibiotics, phages are more host-specific so that the normal microflora would not be destroyed simultaneously when they murder the pathogens. Meanwhile, phages present no cytotoxicity to mammalian cells, and therefore PT shows less side effects on patients compared with antibiotics (Figure 2). Also, the systems that bacteria evolve to negate the antibiotics are not able to defend the phage invadeing, because the phages share different antimicrobial mechanisms with antibiotics. Of course, phages can also be used in many other fields besides phage therapy, such as biocontrol, diagnosis and detection(Pizarro-Bauerle & Ando, 2020).
Figure 2. Tetracycline, an antibiotic used broadly decades ago. Tetracycline was proved to damage teeth and bone. Many other antibiotics are also believed with side effects to human bodies.
However, PT is still not utilized popularly in clinical treatment currently. Due to the narrow host spectrum of the phages and the arms races between phages and their host bacteria, phage cocktails are usually required in PT to guarantee the targeting of all mutant pathogens with various antimicrobial mechanisms(Chan et al., 2013). The main bottlenecks that limit the application of PT are not only the labor-intensive and time-consuming process for preparing phage cocktails which is only affordable in precision medicine and comfort treatment(Chan et al., 2013), but also the great understudies of phage itself, including the functions of some gene sequences and phage-derivative proteins(Brüssow, 2012).
In contrast with the conventional phage therapy strategies, phage genome engieering provides an efficient and specific method to design a novel phage with great functional promotions, it also economize plenty of manpower and material resources that would be spent on phage isolation, purification and characterization. Moreover, manipulations of phage genomes are a trend in PT that engineered phages are thought more controllable and easier to manage, though only one engineered phage preparation has been used clinically(Dedrick et al., 2019). Some non-clinical studies show that the engineered phages represent various novel functions, such as the expansion of host spectrums(Ando et al., 2015a), the destruction of bacterial resistance plasmids and the stabilization of phages in mammalian bodies. As a result, various methodologies for manipulating the phage genome have been developed for both exploring the phage genomes and designing novel phage strains to satisfy clinical requirements. Those strategies include, for example, homologous recombination(Marinelli et al., 2012)(Marinelli et al., 2008), transcription-translation (TXTL) cell-free system(Shin et al., 2012)(Rustad et al., 2017)(Rustad et al., 2018), and yeast artificial chromosome (YAC)-mediated phage reconstruction and rebooting(Jaschke et al., 2012)(Ando et al., 2015a)(Kilcher et al., 2018), yet all of them have various technological limitations. Homologous recombination is a classical strategy to exchange a phage gene fragment with another one with very low efficiency, result in exchanging two or more fragments simultaneously almost an impossible operation. TXTL cell-free system was only reported to synthesize several phages which do not depend on the whole bacterial cells, such as MS2 and T7, yet the mechanism and what kind of phages are able to be assembled in vitro are not clarified(Rustad et al., 2018).
In recent years, YAC-mediated phage reconstruction has developed rapidly, and been becoming the mainstream method for phage engineering, as the this technique provides a platform to de novo edit phage genomes, into which introducing multiple genetic modifications are possible and efficient. Also, active phage particles can be revived via YAC-mediated phage rebooting, which provides a new methodology to obtain phages for therapeutic purposes. Researchers have generated synthetic genomes of some phage species with small genome size(<50kbp), such as coliphage phiX174(Jaschke et al., 2012) and T7(Ando et al., 2015), and K(Gill, 2014) etc. and rebooted them in the host bacteria. In addition, yeast-based assembly platforms wasalso used to construct and engineering animal viruses. for example, the HSV-1(Oldfield et al., 2017). When the terrible pestilence caused by COVID-19 strike the world, utilizing similar YAC platform, researchers successfully obtained live virus particles from the published genome sequence of coronavirus(Thi Nhu Thao et al., 2020).
However, limited by the electroporation efficiency, prokaryotic systems cannot accept a large plasmid as easy as the small plasmids, so that it is technically difficult to manipulate phages with larger genomes (more than 50kbp, also called huge phages) via the classical YAC platform. Given that the median genome size for all the discovered phages is around 52 kb, and most of the strictly lytic phages that are used in PT are huge phages(Al-Shayeb et al., 2020), it’s necessary to optimize the classical YAC platform to edit the genomes of the huge phages.
In our research, we improved the classical YAC-mediated phage rebooting platform to rebuild phage T4, whose genomes are too large (about 168.9 kbp) to manipulate effectively via existing YAC-mediated reconstruction strategy. In brief, inspired by the separation of eukaryotic chromosomes, we divided the phage T4 genome into four huge fragments (40~50kbp per fragments), and each huge fragment was made up with several short gene elements (5~7kbp per element), which were de novo synthesized via gene splicing by overlap extension PCR, so that mutants and exchanges can be introduced in every single element without disturbing the others. Then these gene elements were electroporated into the S. cerevisiae together with the linearized YAC fragments(Ando et al., 2015), and then they are able to connect with each other through CRISPR/Cas9-based mating-type switching(Xie et al., 2018), followed by the amplification in the yeast cells. The huge YAC-phage plasmids, which encoded the whole T4 genome, are introduced into E. coli and the T4 phage will be rebooted.
Figure 3.