Lactococcus lactis is used extensively by the dairy industry worldwide to produce an array of cheeses. Due to its industrial value, this lactic acid bacterium has been widely studied and L. lactis MG1363 is now even a model strain for studies on Gram-positive bacteria. Lactococcal phages are natural inhabitant of milk, survive pasteurization, and pose a significant risk to milk fermentation by infecting L. lactis strains. Virulent lactococcal siphophages belonging to the sk1-like group are by far the most predominant in the dairy industry. Phage p2 is a model for this group and it infects L. lactis MG1363. It has proteinaceous capsid of 69 nm in diameter, which contains its dsDNA genome of 27,595 bp and 49 predicted orfs. Its structural proteins have been analyzed in great details. Still, almost half of its genes encode for proteins of unknown functions. The main reason for this lack of knowledge is the absence of an efficient tool to edit the genome of virulent phages.

Here, we have developed such a tool using the CRISPR-Cas9 technology. This technology is based on a natural anti-viral system, which allows prokaryotes to defend themselves against foreign DNA. Cas9 is an endonuclease that can be programmed to cleave a precise genomic sequence when paired with a CRISPR region, which serves as an RNA guide. When supplied with a DNA template suited for homologous recombination, the host machinery can repair the viral lesion and generate specific mutations. We have adapted CRISPR-Cas9 in L. lactis and started to edit the previously intractable genome of the virulent phage p2. So far, we have inactivated two bona fide non-essential genes, which will allow studying their role in vivo. This tool will enable us to shed light on the numerous phage-encoded proteins that lack any known function.