A phiKMV ligase-dependent DNA repair mechanism that mitigates DNA-targeting nucleases
- 1UCSF
Abstract
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 broadly resistant to multiple CRISPR-Cas and R-M systems. Following CRISPR-Cas12a exposure, most protospacer sequences remained genetically unchanged. However, at an intergenic protospacer, mutations accumulated with high frequency at the Cas12a cleavage site rather than within PAM or seed sequences, resembling repair-associated indels observed after genome editing in eukaryotic cells. Genetic screens to isolate Cas12a- and EcoRI-sensitized phage mutants revealed perturbations to the phage DNA ligase. A Cas12a-sensitive mutant phage was rescued by DNA ligase expression in trans, which was also sufficient to reverse CRISPR targeting of an unrelated phage. Together, our results support a model in which phiKMV-like phages tolerate certain nucleases through ligase-dependent repair of nuclease-induced double-stranded breaks.
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BibTeX
@misc{villani2026phikmv,
title = {{A phiKMV ligase-dependent DNA repair mechanism that mitigates DNA-targeting nucleases}},
author = {A. Villani and S. Karambelkar and N. Pathak and R. Kamath and S. Govindarajan and B. Csorgo and N. Varnavski and J. Bondy-Denomy},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.21.753257},
url = {https://www.biorxiv.org/content/10.64898/2026.09.21.753257v1},
note = {CC BY-NC},
}
RIS
TY - UNPB
TI - A phiKMV ligase-dependent DNA repair mechanism that mitigates DNA-targeting nucleases
AU - A. Villani
AU - S. Karambelkar
AU - N. Pathak
AU - R. Kamath
AU - S. Govindarajan
AU - B. Csorgo
AU - N. Varnavski
AU - J. Bondy-Denomy
PY - 2026
DA - 2026/09/21
PB - bioRxiv
DO - 10.64898/2026.09.21.753257
UR - https://www.biorxiv.org/content/10.64898/2026.09.21.753257v1
AB - 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 broadly resistant to multiple CRISPR-Cas and R-M systems. Following CRISPR-Cas12a exposure, most protospacer sequences remained genetically unchanged. However, at an intergenic protospacer, mutations accumulated with high frequency at the Cas12a cleavage site rather than within PAM or seed sequences, resembling repair-associated indels observed after genome editing in eukaryotic cells. Genetic screens to isolate Cas12a- and EcoRI-sensitized phage mutants revealed perturbations to the phage DNA ligase. A Cas12a-sensitive mutant phage was rescued by DNA ligase expression in trans, which was also sufficient to reverse CRISPR targeting of an unrelated phage. Together, our results support a model in which phiKMV-like phages tolerate certain nucleases through ligase-dependent repair of nuclease-induced double-stranded breaks.
ER -