Genome Engineering of Virulent Lactococcal Phages Using CRISPR-Cas9

Area of Science:

• Microbiology
• Molecular Biology
• Biotechnology

Background:

• Bacteriophages (phages) are ubiquitous biological entities, yet significant knowledge gaps persist regarding their function.
• Engineering virulent lactococcal phages, crucial in dairy fermentation, is hindered by a lack of efficient gene-editing tools.
• Lactococcus lactis is vital for milk fermentation, but its susceptibility to phages threatens the cheese industry.

Purpose of the Study:

• To develop and implement an efficient gene-editing system for modifying virulent lactococcal phages.
• To enable functional studies of previously uncharacterized genes in the lactococcal phage p2 genome.
• To provide a reproducible method for precise genetic manipulation of lytic phages in vivo.

Main Methods:

• Adaptation and implementation of the Streptococcus pyogenes CRISPR-Cas9 system in a heterologous host.
• Application of the CRISPR-Cas9 tool in the phage-sensitive Lactococcus lactis MG1363 host.
• Generation of precise mutations within the genome of the lactococcal phage p2.

Main Results:

• Successful modification of the lactococcal phage p2 genome using the adapted CRISPR-Cas9 system.
• Demonstration of a simple and reproducible technique for generating specific mutations in a virulent phage.
• Facilitation of in vivo studies on lytic phage genes and their encoded proteins.

Conclusions:

• The adapted CRISPR-Cas9 system provides the first efficient gene-editing tool for virulent lactococcal phages.
• This methodology significantly advances the study of phage biology and gene function.
• The technique offers a valuable approach for understanding and potentially controlling phage activity in industrial settings.