CRISPR-Cas9-Mediated Genome Editing in Escherichia coli Bacteriophages

Traditional low-efficiency homologous recombination between plasmids and phage genome was developed to generate recombinant phages with gene replacement, deletion, or insertion. Here, we describe a protocol for well-known Streptococcus pyogenes CRISPR-Cas9-mediated genome editing of lytic Escherichia coli T4 phage. We introduce two methods for point mutation that yield a BamHI restriction site in the target region and a fluorescent protein tag (GFP) insertion in T4 phage genome as examples. Engineered T4 phages were obtained in 100% efficiency. This protocol can be adapted for any other phage modifications by active heterologous CRISPR-Cas9 in their host.