Phage engineering: protection by circularized intermediate

A method for integrating a heterologous nucleic acid into a terminally redundant linear bacteriophage DNA genome comprising:

• (a) cleaving a first site at the 5′ end of a plurality of terminally redundant linear bacteriophage DNA genomes and a second site at the 3′ end of the plurality of terminally redundant linear bacteriophage DNA genomes with a CRISPR enzyme in vitro, wherein the plurality of terminally redundant linear bacteriophage DNA genomes is present in an isolated sample;
• (b) recombining in vitro the cleaved plurality of terminally redundant linear bacteriophage DNA genomes with the heterologous nucleic acid in the presence of a recombination system, wherein the heterologous nucleic acid comprises a 3′ flanking region and a 5′ flanking region that are homologous to the 5′ and 3′ ends of the cleaved plurality of terminally redundant linear bacteriophage DNA genomes respectively, thereby generating a plurality of circularized recombinant bacteriophage DNA genomes;
• and
• (c) enriching the plurality of circularized recombinant bacteriophage DNA genomes by incubating the sample with at least one exonuclease, wherein the heterologous nucleic acid encodes a non-endogenous protein, wherein the plurality of circularized recombinant bacteriophage genomes gives rise to recombinant bacteriophage particles that produce functionally active non-endogenous protein when transformed into a bacterial host cell, wherein the CRISPR enzyme is Cas9, and wherein the plurality of terminally redundant linear bacteriophage DNA genomes are selected from the group consisting of Klebsiella phage K11, Enterobacteria phage T7, and K15 phage.