Researchers at the University of Otago – Ōtākou Whakaihu Waka in New Zealand have developed a CRISPR-based method for systematically disrupting genes across bacteriophage genomes, creating a faster route to uncover gene functions and engineer phages for potential therapeutic and biotechnology applications. The study, titled “Defining the essential genome of diverse phages with phage Tn-seq,” was published in Nature Microbiology.
Bacteriophages, or phages, are viruses that infect bacteria. Their ability to destroy bacterial cells has made them promising candidates for addressing antimicrobial resistance and reducing reliance on agrochemicals. However, many phage genes encode unknown functions, making it difficult to predict how the viruses behave or to modify them reliably.
“Our knowledge of phages is probably like the understanding of antibiotics back in the 1950s,” senior author Peter Fineran, PhD, said. “Many phage genes are currently in the area of microbial dark matter—encoding functions we just don’t understand—which is limiting our ability to use phages in healthcare and biotechnology.”
To address that gap, the team combined transposon insertion sequencing with CRISPR–anti-CRISPR selection. A transposon jumps into a phage genome and disrupts a gene. The selection system then recovers phages carrying those mutations, allowing researchers to determine which genes can be disrupted without preventing the virus from surviving and which are essential for phage function.
After establishing the mutagenesis workflow, the researchers extended it to introduce new genetic cargo. Rather than using the transposon only to disrupt genes, they loaded it with an additional sequence and inserted a fluorescent marker into phage genomes. The approach could also be used to add genes that help phages overcome bacterial defense systems, potentially improving their effectiveness against target pathogens.
“Once we had that established, we thought we could expand the technique and also use it to add genes directly into the phage genomes,” co-lead author Manuela Fuchs, PhD, said.
“This is a systematic, broadly applicable, and cost-effective way to not only investigate gene function, but also rapidly engineer phage genomes,” senior author Leah Smith, PhD, said, in a statement.
The platform could therefore serve two related goals: mapping the largely uncharacterized genetic landscape of phages and building variants with new capabilities. The researchers said the work opens opportunities for fundamental studies as well as future therapeutic development, although engineered phages would still require further testing to establish their safety and effectiveness in specific applications.