New CRISPR-Based Tool Mutates and Engineers Bacteriophage Genomes

Researchers at the University of Otago have developed a new method for mutating and engineering bacteriophages, the viruses that infect and destroy bacteria. 

Bacteriophages, or phages, are seen as a promising tool against the antimicrobial resistance crisis and as a more sustainable alternative to agrochemicals in agriculture, because of their ability to destroy bacteria. But, according to Professor Peter Fineran, senior author of new research published in Nature Microbiology, the field’s grasp of how phage genes actually work remains limited. “But our knowledge of phages is probably like the understanding of antibiotics back in the 1950s. 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.” He describes the new method as “a big leap in how we can rapidly understand phage biology and then use that knowledge to make phages work better to help achieve our goals.”

Co-lead author Manuela Fuchs says the technique enables genome-wide mutagenesis of bacteriophages using CRISPR-Cas technology. The team used transposon insertion sequencing, in which a mobile piece of DNA jumps into and disrupts a gene, combined with CRISPR-anti-CRISPR-based selection to isolate mutated phages. Together, these tools let the researchers distinguish genes that are essential for phage survival from those that are not.

Fuchs says the team then found the transposon could carry more than the anti-CRISPR protein used for selection. “Once we had that established, we thought we could expand the technique and also use it to add genes directly into the phage genomes. We essentially found it is possible to load additional genes on that transposon, not just our anti-CRISPR protein. While we added a fluorescent marker, it could be used to, for example, add additional anti-defense genes to phages, to improve their therapeutic potential.”

Senior author Leah Smith says the method inserts genes into the phage genome while preserving phage function. “This is a systematic, broadly applicable, and cost-effective way to not only investigate gene function, but also rapidly engineer phage genomes. This opens up new opportunities for both fundamental research and future therapeutic development.”

Smith points to biofilms, which drive hard-to-treat infections on prosthetic implants and medical devices, as one potential application. “With this new technique, we could quickly load phages to have more things that stop some bacterial defenses so that they can be harnessed to kill pathogens more easily.”