LAUSR

Tools for site-directed mutagenesis of virulent bacteriophages (phages; viruses of bacteria) have traditionally lagged those for bacteria, hindering their study. CRISPR gene editing represents a new and highly efficient method for editing virulent phage genomes. Here, I describe methods for using CRISPR gene editing for site-directed mutagenesis of ICP1, a virulent phage of Vibrio cholerae The first section outlines methods of constructing a plasmid for CRISPR editing of an ICP1 gene. The second section outlines methods of transferring the plasmid to an editing-competent strain of V. cholerae The third section outlines methods of selecting for and storing the edited phage.