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ACS Synth Biol. 2017 Jul 21;6(7):1351-1358. doi: 10.1021/acssynbio.6b00388. Epub 2017 Mar 30.

Genome Engineering of Virulent Lactococcal Phages Using CRISPR-Cas9.

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Département de biochimie, de microbiologie, et de bioinformatique, Faculté des sciences et de génie, Félix d'Hérelle Reference Center for Bacterial Viruses, and Groupe de recherche en écologie buccale, Faculté de médecine dentaire, Université Laval , Québec City, Québec G1V 0A6, Canada.


Phages are biological entities found in every ecosystem. Although much has been learned about them in past decades, significant knowledge gaps remain. Manipulating virulent phage genomes is challenging. To date, no efficient gene-editing tools exist for engineering virulent lactococcal phages. Lactococcus lactis is a bacterium extensively used as a starter culture in various milk fermentation processes, and its phage sensitivity poses a constant risk to the cheese industry. The lactococcal phage p2 is one of the best-studied models for these virulent phages. Despite its importance, almost half of its genes have no functional assignment. CRISPR-Cas9 genome editing technology, which is derived from a natural prokaryotic defense mechanism, offers new strategies for phage research. Here, the well-known Streptococcus pyogenes CRISPR-Cas9 was used in a heterologous host to modify the genome of a strictly lytic phage. Implementation of our adapted CRISPR-Cas9 tool in the prototype phage-sensitive host L. lactis MG1363 allowed us to modify the genome of phage p2. A simple, reproducible technique to generate precise mutations that allow the study of lytic phage genes and their encoded proteins in vivo is described.


CRISPR-Cas9; double-strand DNA break repair; genome engineering; homologous recombination; lactic acid bacteria; virulent bacteriophages

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