CRISPR Gene Editing of Virulent Bacteriophage ICP1

Abstract

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.

FIGURE 1.

Illustration of the plasmid pCRISPR (Box et al. 2015) used for CRISPR gene editing in Vibrio cholerae. Two BsaI restriction sites flank a removable spacer located within a mini-CRISPR array. The two sets of overhangs generated by BsaI digestion differ from each other, allowing for directional cloning of a new spacer into the mini-CRISPR array. Other relevant features of pCRISPR include the lacIq gene, which controls IPTG-inducible expression of the P_tac promoter and thus the downstream mini-CRISPR array, a bla gene encoding carbenicillin resistance, and oriT for conjugative transfer.