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Nucleic Acids Res. 2014 Aug;42(14):9504-13. doi: 10.1093/nar/gku628. Epub 2014 Jul 24.

CRISPR-Cas: an efficient tool for genome engineering of virulent bacteriophages.

Author information

1
Département de biochimie, microbiologie et bio-informatique, Faculté des sciences et de génie, Université Laval, Quebec city, Quebec, G1V 0A6, Canada Groupe de recherche en écologie buccale, Faculté de médecine dentaire, Université Laval, Quebec City, Quebec, G1V 0A6, Canada.
2
Département de biochimie, microbiologie et bio-informatique, Faculté des sciences et de génie, Université Laval, Quebec city, Quebec, G1V 0A6, Canada Groupe de recherche en écologie buccale, Faculté de médecine dentaire, Université Laval, Quebec City, Quebec, G1V 0A6, Canada Sylvain.Moineau@bcm.ulaval.ca.

Abstract

Bacteriophages are now widely recognized as major players in a wide variety of ecosystems. Novel genes are often identified in newly isolated phages as well as in environmental metavirome studies. Most of these novel viral genes have unknown functions but appear to be coding for small, non-structural proteins. To understand their biological role, very efficient genetic tools are required to modify them, especially in the genome of virulent phages. We first show that specific point mutations and large deletions can be engineered in the genome of the virulent phage 2972 using the Streptococcus thermophilus CRISPR-Cas Type II-A system as a selective pressure to increase recombination efficiencies. Of significance, all the plaques tested contained recombinant phages with the desired mutation. Furthermore, we show that the CRISPR-Cas engineering system can be used to efficiently introduce a functional methyltransferase gene into a virulent phage genome. Finally, synthetic CRISPR bacteriophage insensitive mutants were constructed by cloning a spacer-repeat unit in a low-copy vector illustrating the possibility to target multiple regions of the phage genome. Taken together, this data shows that the CRISPR-Cas system is an efficient and adaptable tool for editing the otherwise intractable genomes of virulent phages and to better understand phage-host interactions.

PMID:
25063295
PMCID:
PMC4132740
DOI:
10.1093/nar/gku628
[Indexed for MEDLINE]
Free PMC Article

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