Bacteriophages likely constitute
the largest biomass on Earth.
However, very few phage genomes have been well-characterized, the
tailed phage T4 genome being one of them. Even in T4, much of the
genome remained uncharacterized. The classical genetic strategies
are tedious, compounded by genome modifications such as cytosine hydroxylmethylation
and glucosylation which makes T4 DNA resistant to most restriction
endonucleases. Here, using the type-II CRISPR-Cas9 system, we report
the editing of both modified (ghm-Cytosine) and unmodified (Cytosine)
T4 genomes. The modified genome, however, is less susceptible to Cas9
nuclease attack when compared to the unmodified genome. The efficiency
of restriction of modified phage infection varied greatly in a spacer-dependent
manner, which explains some of the previous contradictory results.
We developed a genome editing strategy by codelivering into <i>E. coli</i> a CRISPR-Cas9 plasmid and a donor plasmid containing
the desired mutation(s). Single and multiple point mutations, insertions
and deletions were introduced into both modified and unmodified genomes.
As short as 50-bp homologous flanking arms were sufficient to generate
recombinants that can be selected under the pressure of CRISPR-Cas9
nuclease. A 294-bp deletion in RNA ligase gene <i>rnlB</i> produced viable plaques, demonstrating the usefulness of this editing
strategy to determine the essentiality of a given gene. These results
provide the first demonstration of phage T4 genome editing that might
be extended to other phage genomes in nature to create useful recombinants
for phage therapy applications