TY  - JOUR
TI  - Targeted Genome Editing of Virulent Phages Using CRISPR-Cas9
AU  - Lemay, Marie-Laurence 
AU  - Renaud, Ariane 
AU  - Rousseau, Geneviève M.
AU  - Moineau, Sylvain  
VL  - 8
IS  - 1
PY  - 2018
DA  - 2018/01/05
SP  - e2674
C1  - Bio-protocol 2018;8:e2674
DO  - 10.21769/BioProtoc.2674
UR  - https://doi.org/10.21769/BioProtoc.2674
AB  - This protocol describes a straightforward method to generate specific mutations in the genome of strictly lytic phages. Briefly, a targeting CRISPR-Cas9 system and a repair template suited for homologous recombination are provided inside a bacterial host, here the Gram-positive model Lactococcus lactis MG1363. The CRISPR-Cas9 system is programmed to cleave a specific region present on the genome of the invading phage, but absent from the recombination template. The system either triggers the recombination event or exerts the selective pressure required to isolate recombinant phages. With this methodology, we generated multiple gene knockouts, a point mutation and an insertion in the genome of the virulent lactococcal phage p2. Considering the broad host range of the plasmids used in this protocol, the latter can be extrapolated to other phage-host pairs.
KW  - Phage
KW  - Lactococcus lactis
KW  - Genome editing
KW  - CRISPR-Cas9
KW  - Homologous recombination
JF  - Bio-protocol
SN  - 2331-8325
PB  - Bio-protocol LLC.
BIO101  - False