Utilizing Golden Gate Assembly to Streamline CRISPR-Cas/NgTET-Based Phage Mutagenesis.

Phage engineering is an emerging technology due to the promising potential application of phages in medical and biotechnological settings. Targeted phage mutagenesis tools are required to customize the phages for a specific application and generate, in addition to that, so-called designer phages. CRISPR-Cas technique is used in various organisms to perform targeted mutagenesis. Yet, its efficacy is notably limited for phage mutagenesis due to the highly abundant phage DNA modifications. Addressing this challenge, we have developed a novel approach that involves the temporal removal of phage DNA cytosine modifications, allowing for effective CRISPR-Cas targeting and subsequent introduction of mutations into the phage genome. The removal of cytosine modification relies on the catalytic activity of a eukaryotic ten-eleven translocation methylcytosine (TET) dioxygenase. TET enzymes iteratively de-modify methylated or hydroxymethylated cytosines on phage DNA. The temporal removal of cytosine modification ultimately enables efficient DNA cleavage by Cas enzymes and facilitates mutagenesis. To streamline the application of the coupled TET-CRISPR-Cas system, we use Golden Gate cloning for fast and efficient assembly of a vector that comprises a TET oxidase and a donor DNA required for scarless site-specific phage mutagenesis. Our approach significantly advances the engineering of modified phage genomes, enabling the efficient generation of customized phages for specific applications.