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