A phiKMV ligase-dependent DNA repair mechanism that mitigates DNA-targeting nucleases
Bacteria employ diverse DNA-targeting systems, including restriction-modification (R-M) and CRISPR-Cas, to cleave invading bacteriophage genomes. In response, phages encode counter-defense strategies that block or mitigate DNA damage. Here, we screened a panel of Pseudomonas aeruginosa phages against native and heterologous DNA-targeting systems and identified the Phikmvvirus phage genus as…
Bacteria possess a variety of DNA-targeting mechanisms, such as restriction-modification and CRISPR-Cas, to defend against invading phage genomes. In response to these threats, phages develop countermeasures to either block or alleviate DNA damage. Researchers screened a collection of Pseudomonas aeruginosa phages against native and foreign DNA-targeting systems, discovering that the Phikmvvirus phage genus displayed broad resistance to multiple CRISPR-Cas and restriction-modification systems.
After exposure to CRISPR-Cas12a, the majority of protospacer sequences maintained their genetic integrity. However, at an intergenic region adjacent to the CRISPR-Cas12a cleavage site, mutations emerged at a higher frequency than within the PAM or seed sequences, akin to repair-associated indels observed following genome editing in eukaryotic cells.
Genetic screening efforts aimed at isolating Cas12a- and EcoRI-sensitive phage mutants uncovered disruptions in the phage DNA ligase. A phage strain resistant to Cas12a was restored by introducing DNA ligase from a different genetic source, and this rescue effect also reversed CRISPR targeting in a non-related phage. The findings suggest a mechanism whereby phiKMV-like phages manage to withstand specific nucleases through ligase-mediated repair of double-stranded breaks induced by nucleases.
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