Selection of Genetically Modified Bacteriophages Using the CRISPR-Cas System.

Abstract

We present a CRISPR-Cas based technique for deleting genes from the T7 bacteriophage genome. A DNA fragment encoding homologous arms to the target gene to be deleted is first cloned into a plasmid. The T7 phage is then propagated in Escherichia coli harboring this plasmid. During this propagation, some phage genomes undergo homologous recombination with the plasmid, thus deleting the targeted gene. To select for these genomes, the CRISPR-Cas system is used to cleave non-edited genomes, enabling isolation of the desired recombinant phages. This protocol allows seamless deletion of desired genes in a T7 phage, and can be expanded to other phages and other types of genetic manipulations as well.