A team led by Dorentina Humolli and Alexander Harms at ETH Zürich has now introduced a method designed to close this gap. Writing in Nature Microbiology, the researchers present HIDEN-SEQ, short for hidden Acr-enabled transposon-insertion sequencing, a genome-wide approach that systematically links viral genes to measurable phenotypes. The technique adapts transposon-insertion sequencing, or TnSeq, a method that revolutionized bacterial genetics by quantifying the fitness consequences of thousands of random insertions across a genome through comparative deep sequencing. Until now, no broadly applicable equivalent existed for phages, largely because transposition into viral genomes was inefficient and lacked a general selection marker for identifying phage clones that had acquired an insertion.

HIDEN-SEQ solves this problem with an elegant two-part design. First, the mariner transposon Himar1, which inserts randomly at TA dinucleotide sites, was engineered to carry an anti-CRISPR gene, acrVIA1, under a strong constitutive promoter. Second, the bacterial host expresses the CRISPR effector LseCas13a together with a guide RNA targeting the transcripts of an essential phage gene, the major capsid protein. Only phage clones that have picked up the transposon, and therefore express the anti-CRISPR protein, can evade Cas13a targeting and replicate. The choice of a type VI CRISPR system is technically significant: because Cas13a recognizes and cleaves RNA rather than DNA, it bypasses the DNA-based genome protection mechanisms that tailed phages typically deploy, and its collateral cleavage of host and viral RNA aborts infection of any unprotected phage. The name HIDEN-SEQ reflects this molecular game of hide and seek between host-expressed Cas13a and the transposon-encoded anti-CRISPR protein.

To benchmark the method, the team applied it to phage T4, the classic model virus with a 168-kilobase genome encoding nearly 300 genes, around 130 of which remain uncharacterized despite decades of study. The input library achieved 97.2 percent saturation of TA sites across the genome, confirming highly efficient transposition. After outgrowth, insertions in essential genes were strongly depleted, and the analysis concluded that 190 T4 genes are dispensable for infection of the laboratory strain Escherichia coli K-12 under standard conditions, in close agreement with the essentiality map assembled over decades of prior research. Only a handful of genes appeared falsely essential, a discrepancy the authors attribute largely to polar effects, the disruption of neighboring gene expression by an inserted element. Notably, they deliberately omitted a transcriptional terminator from the transposon to minimize this artifact, and showed experimentally that adding one indeed caused strong polar effects.

The resolution of the method extends beyond binary essential or non-essential calls. Genes could be classified as showing weakly, moderately or strongly reduced fitness when disrupted, and in some cases the data resolved essentiality at subgene level. The T4 RNA ligase gene rnlA, for example, tolerated insertions only in its C-terminal domain, consistent with earlier work showing that this region directs the enzyme toward tRNA breaks in the anticodon loop. This depth of information transforms a simple viability screen into a nuanced fitness atlas of the viral genome.

The most striking application of HIDEN-SEQ lies in identifying conditionally essential genes, those required only under specific challenges. When the T4 library was propagated on hosts expressing particular bacterial defence systems, insertions in the corresponding antidefence genes were selectively depleted. The known antidefence genes rIIA and rIIB, which counteract the RexAB system, and dmd, an inhibitor of the RnlAB toxin-antitoxin system, behaved exactly as expected. The screen also recovered the anti-CBASS protein Acb1 and the anti-DarTG1 factor AdfN. Crucially, when the library was challenged with the DarTG2 defence system, a single uncharacterized gene, tk.4, showed specific depletion. The encoded protein is homologous to the DarG antitoxin of Mycobacterium tuberculosis and carries a macrodomain typical of ADP-ribosylglycohydrolases, leading the team to name it AdfM, a previously unknown anti-DarTG2 factor that probably removes ADP-ribose modifications from viral DNA.

Conditional essentiality was not limited to immunity. Comparing the library grown in rich Lysogeny Broth with growth in defined M9 minimal medium revealed that the T4 gene vs, encoding a modifier of valyl-tRNA synthetase, was the most strongly depleted under nutrient limitation, and a deletion mutant indeed failed to form plaques efficiently in minimal medium. This demonstrates that HIDEN-SEQ can expose genes needed for phage replication across different physiological states of the host, not only in the context of antiviral defence.

To test portability, the researchers applied the method to two further E. coli phages. Bas37, a close relative of T4, yielded essentiality patterns broadly similar to T4, with informative exceptions: its RNA ligase required both protein domains, and the regA orthologue, a translational repressor, was essential in Bas37 but dispensable in T4, differences validated by targeted gene disruptions. Bas54, a member of the poorly studied Vequintavirinae with 227 predicted genes, proved equally amenable, achieving 91.6 percent TA-site saturation. In Bas54, 145 genes were non-essential, several bona fide tail fibre genes tolerated disruptive insertions, and remarkably, 15 of the 44 essential genes lacked any functional annotation, underscoring how much of the essential phage genome remains obscure even beyond classical models.

The team then pushed the approach into a clinically relevant setting by infecting a panel of uropathogenic E. coli isolates with the T4, Bas37 and Bas54 libraries. For each phage-host pair, multiple viral genes showed strongly reduced insertion counts relative to the laboratory strain, marking candidate antidefence factors. Using DefenseFinder to predict defence systems in the clinical genomes, followed by targeted deletions of the most depleted phage genes and ectopic expression of individual defence systems in E. coli K-12, the researchers matched several candidates to specific branches of bacterial immunity. Among the findings, the Alt ADP-ribosyltransferase of T4 and a beta-alpha glucosyltransferase of Bas37 were implicated in evading the Mokosh type I defence system, with only the T4 version of Alt able to partially restore plaque formation when expressed in trans. Two previously uncharacterized proteins, T4 NrdC.1 and Bas37_0203, were confirmed as antidefence factors against the Septu and Druantia type III systems respectively, despite lacking any known domains or homologues. Bas37_0260, which shares no homology with T4’s type IV restriction inhibitors beyond a conserved N-terminal signal sequence, proved to be a novel inhibitor of McrBC. In Bas54, a MoxR ATPase and a gene of unknown function were both required to overcome the predicted DS-28 defence system of one clinical isolate.

Taken together, HIDEN-SEQ establishes a versatile platform that connects phage phenotypes to underlying genes and molecular mechanisms. Compared with recently developed alternatives such as knockdown-based CRISPRi approaches, which failed to detect the essentiality of roughly a quarter of known essential T4 genes, or targeted mutagenesis methods that require extensive custom construct design, HIDEN-SEQ demands no prior knowledge of the target phage’s genes, relies on a single guide RNA for selection, and produces high-density insertion libraries that can be reused across many hosts and conditions. The authors note that related transposon-sequencing approaches reported for phages of Pseudomonas and Prodigiosinella during the study’s revision confirm the broad applicability of phage transposon mutagenesis, while suggesting that individual viruses may require tailored adjustments such as phage-specific promoters or alternative transposon systems to overcome DNA modifications. With its capacity to reveal dispensable genome regions useful for phage engineering and to assign functions to the dark matter of viral genomes, HIDEN-SEQ promises to accelerate progress in phage therapy, microbial ecology and biotechnology at a moment when new weapons against antibiotic-resistant bacteria are urgently needed.

Subject of Research: A transposon-insertion sequencing method for mapping bacteriophage gene essentiality and discovering antidefence genes

Article Title: Systematic mapping of bacteriophage gene essentiality with HIDEN-SEQ

Article References: Humolli, D., Piel, D., Ransome, J., Bausch, K., Tschudin-Sutter, S., Ortelli, M., Dehio, C., Veening, J.-W., & Harms, A. (2026). Systematic mapping of bacteriophage gene essentiality with HIDEN-SEQ. Nature Microbiology. https://doi.org/10.1038/s41564-026-02455-8

Image Credits: AI Generated

DOI: 10.1038/s41564-026-02455-8

Keywords: bacteriophages, HIDEN-SEQ, transposon-insertion sequencing, gene essentiality, phage therapy, CRISPR-Cas13a, anti-CRISPR, antidefence genes, phage T4, functional genomics, bacterial defence systems, microbiology

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

The centerpiece of the study is an in vivo CRISPR interference, or CRISPRi, screening platform. Unlike traditional knockouts, which permanently delete genes and can be impractical at genome-wide scale, CRISPRi uses a catalytically dead Cas9 protein, dCas9, guided by hundreds of thousands of small RNA molecules to reversibly silence transcription of individual genes. Each bacterial cell in a massive pooled library carries a guide RNA targeting a specific gene, so the abundance of each variant after a period in the host serves as a direct readout of how important the silenced gene was for survival. If a gene is essential in the gut, cells whose guide suppresses it will dwindle in number; if it is dispensable, those cells persist. Gene fitness, measured by deep sequencing of guide RNA abundance with unique molecular identifiers to reduce noise, thus becomes a high-resolution functional reporter of adaptation.

To make the experiment tractable, the researchers colonized mice with OligoMM12, a defined minimal microbial community of twelve mouse gut strains. This gnotobiotic approach, building on earlier genome-guided designs of minimal microbiotas, provides a reproducible yet realistic ecological backdrop in which E. coli must compete with other residents for nutrients and space, without the overwhelming complexity of a conventional microbiome. The team introduced genome-wide CRISPRi libraries into three very different E. coli strains: MG1655, the classic laboratory K-12 derivative; CFT073, a uropathogenic strain; and LF82, an adherent-invasive E. coli, or AIEC, associated with Crohn’s disease. As the libraries circulated through the mice, sequencing revealed which genes each strain depended on under specific conditions.

The first major finding concerns diet. When mice were fed standard chow, a high-fiber diet, or a high-fat Western-style diet, the metabolic landscape facing E. coli changed dramatically, and the essential gene profile shifted with it. Genes required for growth on one diet were dispensable or even deleterious on another, confirming at functional-genomic resolution that what an animal eats directly rewrites the nutritional rules of the gut. The screen also exposed cross-feeding interactions, identifying genes whose loss was tolerated only because other members of the defined community supplied the missing metabolites. In this way the platform does more than list essential genes; it sketches the metabolic web that connects E. coli to its microbial neighbors, showing that the bacterium’s survival strategy is woven into the ecology of the whole community.

Comparing the three strains side by side revealed that there is no single genetic playbook for intestinal colonization. Although the core essential genomes overlapped, reflecting shared housekeeping functions, each strain displayed a distinct signature of requirements. The analysis highlighted divergent strategies in motility, stress response and respiration. MG1655, CFT073 and LF82 each relied on different subsets of genes to cope with bile, acidity, oxygen tension and immune pressures, consistent with the idea that genetic diversity across the E. coli species translates into fundamentally different ecological tactics. The results extend earlier transposon-insertion sequencing studies in enterohemorrhagic E. coli, K1 strains and other pathogens, but with the added precision and tunability that CRISPRi offers, since partial silencing can reveal dose-sensitive phenotypes that complete knockouts miss.

One of the most medically relevant strands of the study concerns AIEC LF82, a strain strongly enriched in the ileal mucosa of Crohn’s disease patients. When the researchers induced intestinal inflammation with dextran sulfate sodium, they found that LF82 preferentially colonized the small intestine in the inflammatory environment, and its genetic dependencies shifted accordingly. Genes tied to iron acquisition became more important, echoing the known biology of lipocalin 2 and neutrophil gelatinase-associated lipocalin, host proteins that sequester iron-bearing siderophores during inflammation. Respiratory genes also rose in prominence, in line with evidence that inflammation disrupts intestinal oxygen balance and creates new terminal electron acceptors that facultative anaerobes can exploit. The inflammatory gut, in other words, is a different nutritional world, and the screen captured that transformation in genetic terms.

Perhaps the most striking discovery involves a mobile genetic element. LF82 carries the Gally prophage, and the CRISPRi data revealed that this integrated virus plays an important role in modulating the strain’s fitness in the inflamed gut. Follow-up analyses quantified phage particle release and showed that the prophage’s influence on fitness differed between inflamed and healthy conditions, adding to growing evidence that prophages are not merely parasitic passengers but functional modulators of host physiology. Previous work has shown that phage integration can rewire the respiratory strategy of E. coli, and the new findings place the Gally element squarely within the colonization toolkit of a Crohn’s-associated pathobiont. The observation that phage production itself appears to be regulated, and blocked under some host-like conditions, underscores how finely tuned these relationships are.

Technically, the study sets a new benchmark for rigor. Gene-level calls were derived using MAGeCK with robust ranking aggregation and Benjamini-Hochberg false-discovery-rate correction across individual mice as biological replicates, and guide activity predictions were used to improve on-target performance, drawing on earlier CRISPR-dCas9 screening work in E. coli. Raw CRISPRi and 16S sequencing data are available through the Sequence Read Archive under accession PRJNA1293909, and processed log2 fold-change values, statistical results, analysis scripts and notebooks are shared through public repositories, allowing other groups to reproduce and extend the work. The authors acknowledge support from the European Research Council, the Agence Nationale de la Recherche, the Fondation pour la Recherche Médicale and the Swiss National Science Foundation’s NCCR AntiResist program.

The broader significance of the work extends well beyond E. coli. By demonstrating that gene fitness can serve as a quantitative sensor of the gut environment, the platform offers a way to probe intestinal conditions directly through the organisms that live there, effectively turning bacteria into living microsensors of diet, inflammation and microbial competition. For microbiome science, where correlational studies abound, this is a shift toward mechanism: every essential gene identified is a hypothesis about a nutrient, a stress or a host defense that can be tested. For medicine, the strain-specific genetic requirements of Crohn’s-associated AIEC and the role of the Gally prophage suggest new angles for interventions that disarm pathobionts without indiscriminately wiping out beneficial residents. As researchers refine gnotobiotic models and CRISPRi delivery, in vivo functional genomics of this kind could become a routine tool, mapping not just which microbes are present in the gut but precisely which molecular functions make them winners or losers in one of the most densely populated ecosystems in the mammalian body.

Subject of Research: In vivo CRISPRi functional genomics of Escherichia coli adaptation to the mouse gut

Article Title: In vivo CRISPRi screens reveal Escherichia coli functional adaptations in the mouse gut

Article References: Maire, A., Ortelli, M., Tkacz, E., Dehio, C., Chassaing, B., Sokol, H., Rolhion, N., & Bikard, D. (2026). In vivo CRISPRi screens reveal Escherichia coli functional adaptations in the mouse gut. Nature Microbiology. https://doi.org/10.1038/s41564-026-02471-8

Image Credits: AI Generated

DOI: 10.1038/s41564-026-02471-8

Keywords: E. coli, CRISPRi, gut microbiome, Nature Microbiology, adherent-invasive E. coli, Crohn's disease, Gally prophage, gene essentiality, mouse model, OligoMM12, functional genomics, intestinal colonization