Precise Phage Mutagenesis with NgTET-Assisted CRISPR-Cas Systems

Bacteriophages, viruses that specifically target their bacterial hosts, hold significant potential for biotechnology and medicine, especially in combating multidrug-resistant infections. However, the molecular mechanisms underlying phage infection remain largely underexplored. Precise, site-specific mutagenesis of phages is a powerful tool to elucidate gene functions and phage-host interactions. However, a major challenge in phage genome mutagenesis is the presence of phage DNA modifications that interfere with conventional genome editing tools like CRISPR-Cas. While CRISPR-Cas systems have been used successfully for targeted mutagenesis in various organisms, their effectiveness in phage mutagenesis is often limited by DNA modifications such as cytosine glycosylation. To overcome this barrier, we developed an efficient method that temporarily reduces the abundance of phage DNA modifications, enabling efficient CRISPR-Cas targeting and precise mutation introduction into phage genomes. Specifically, we use the Ten Eleven Translocation (TET) methylcytosine dioxygenase from Naegleria gruberi (NgTET), which iteratively demodifies methylated and hydroxymethylated cytosines in DNA. By oxidizing hydroxymethylated cytosines within phage DNA, NgTET prevents subsequent cytosine modification like glycosylation and significantly enhances the efficiency of Cas-mediated DNA cleavage. In conclusion, the scarless and precise genome-editing approach presented here enables the efficient introduction of point mutations while maintaining the native gene architecture in phage genomes. By preserving intact transcriptional and translational frameworks, this method minimizes unintended disruptions to complex regulatory networks. This is particularly important for investigating essential or multifunctional phage proteins. The ability to generate targeted genetic modifications without introducing extraneous sequences significantly expands the experimental toolkit for phage biology. This strategy not only facilitates detailed functional studies but also enhances the potential for rational engineering of phages for therapeutic and biotechnological applications.