The central challenge the researchers tackled is one of scale. Traditional phage genetics proceeds gene by gene: a researcher deletes or mutates a candidate gene and observes the consequence. That approach is slow, biased toward genes with predictable functions, and poorly suited to the many phage genes of unknown purpose that dominate most viral genomes. Transposon-insertion sequencing, or Tn-seq, has transformed bacterial functional genomics by creating saturated libraries of mutants and reading out insertion sites with deep sequencing, but adapting the technique to phages has been difficult because phages cannot be transformed with plasmids and their replication cycles are brief and tightly regulated.

The Otago team solved the selection problem with an elegant trick borrowed from the ongoing evolutionary arms race between bacteria and viruses. Their system couples Tn5 transposon mutagenesis to anti-CRISPR-based counter-selection. Phages are first propagated in host bacteria that express the Tn5 transposase together with a transposon carrying an anti-CRISPR gene, in this case AcrVIA1, which inhibits the RNA-targeting nuclease Cas13a. Only phage genomes that have acquired the transposon, and therefore the anti-CRISPR gene, can replicate in a second host strain that expresses Cas13a loaded with spacers targeting the phage. Unmutated phages are destroyed or blocked by the CRISPR defense, while transposon-bearing mutants are selectively enriched. Deep sequencing of the enriched population then reveals, across the whole genome, where insertions survived and where they were eliminated.

The logic of the readout is straightforward: genes that tolerate no transposon insertions after enrichment are essential for phage replication, whereas genes riddled with insertions are dispensable. Applying the method to phage JS26, a T7-like virus of the bacterium Prodigiosinella confusarubida, the researchers generated a genome-wide essentiality map that agreed well with independent lines of evidence, including structural proteomics of purified virions and conservation of core genes across related phages. Genes encoding capsid and tail components, DNA replication machinery, and other core functions showed the expected depletion of insertions, while genes of unknown or auxiliary function were heavily disrupted.

A particularly demanding test was the nucleus-forming jumbo phage PCH45, a relative of the well-studied phiKZ-like viruses of Pseudomonas. These giant phages, with genomes exceeding 200 kilobases, build a nucleus-like proteinaceous compartment inside the infected cell that shields their replicating DNA from CRISPR-Cas nucleases. The team found that standard transposase worked poorly in this context, so they engineered a fusion between the transposase and UvsX, a phage recombination protein that localizes to the viral nucleus. This UvsX-TnpA fusion dramatically improved mutagenesis of PCH45, allowing the researchers to construct the first genome-scale essentiality map for a nucleus-forming jumbo phage and to distinguish essential core genes from the large complement of non-essential genes that characterize these remarkable viruses.

Beyond essentiality, the method extracts information from the biases inherent in transposon insertion. Because Tn5 inserts preferentially into certain sequence contexts and strand orientations, the distribution of insertions carries signals about genome organization. The researchers showed that insertion orientation biases allowed them to predict the direction of transcription of phage genes, and that insertion density correlated with expression levels, enabling identification of regions injected early into the host and genes expressed at high levels during infection. The insertion index across non-essential genes showed a moderate positive correlation with RNA-seq expression measurements, demonstrating that a single mutagenesis experiment can yield both functional and regulatory information.

The transposon itself proved to be a versatile delivery vehicle. By modifying the cargo carried within the transposon, the team generated phages labeled with fluorescent proteins within a few days, a task that conventional phage engineering approaches can take weeks to accomplish. They also created an orthogonal system based on an artificial intelligence-designed anti-CRISPR protein, allowing a second, distinguishable transposon to be delivered into phages that already carried the first. Using this approach they constructed phage double mutants carrying two different transposons, each conferring resistance to a different Cas13a variant, which permitted sequential counter-selection. The strategy worked across several diverse phages, including LC53 and Bas46, infecting different bacterial hosts.

One further technical hurdle was DNA modification. Many phages protect their genomes from host defenses with heavily modified bases, such as hypermodified cytosines, which can block enzymatic steps in genome engineering. The researchers demonstrated that transposon insertion was achievable even in phages with such hypermodified DNA, extending the reach of the method to viruses whose chemical biology has historically resisted manipulation. This breadth matters because modified DNA is common among phages that are attractive candidates for therapeutic development, precisely because modifications help them evade bacterial immune systems.

The implications extend in two directions. For basic science, phage Tn-seq provides an unbiased, high-throughput entry point into the functional genomics of viruses whose genes are largely unannotated, complementing recent parallel efforts such as CRISPRi-based phage screens and other transposon approaches reported for bacteriophages. Essentiality maps anchored by proteomics and conservation data give researchers a rational basis for assigning functions to the growing torrent of phage genome sequences generated by environmental sequencing. For applied science, the ability to insert cargo rapidly and at many genomic positions addresses a persistent bottleneck in phage therapy engineering, where researchers seek to equip therapeutic phages with traits such as reporter genes, altered host ranges, or additional anti-defense functions without laborious homologous recombination protocols.

The authors have made the underlying resources broadly available. Raw sequencing data are deposited in the NCBI Sequence Read Archive under BioProject PRJNA1381746, proteomics datasets are accessible through the PRIDE repository, and the plasmids developed for the system, including the transposon delivery and selection constructs, are deposited at Addgene. As phage-based antimicrobials move closer to clinical and agricultural deployment amid rising antibiotic resistance, tools that compress the design-build-test cycle for viral genomes are likely to become standard equipment in the phage engineering toolbox. Phage Tn-seq, by simultaneously mapping what a phage cannot live without and delivering what engineers want to add, offers both the map and the vehicle in a single experiment.

Subject of Research: Genome-wide essentiality mapping and transposon-based engineering of bacteriophages

Article Title: Defining the essential genome of diverse phages with phage Tn-seq

Article References: Kyte, N., Fuchs, M., Smith, L. M., & Fineran, P. C. (2026). Defining the essential genome of diverse phages with phage Tn-seq. Nature Microbiology. https://doi.org/10.1038/s41564-026-02486-1

Image Credits: AI Generated

DOI: 10.1038/s41564-026-02486-1

Keywords: bacteriophage, phage Tn-seq, transposon mutagenesis, gene essentiality, anti-CRISPR, jumbo phage, phage engineering, functional genomics, phage therapy, Cas13a, DNA modification, biotechnology