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Published November 22, 2021 | Submitted + Supplemental Material
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Genome manipulation by guide-directed Argonaute cleavage

  • 1. ROR icon California Institute of Technology


Many prokaryotic argonautes (pAgos) mediate DNA interference by using small DNA guides to cleave target DNA. A recent study shows that CbAgo, a pAgo from Clostridium butyricum, induces DNA interference between homologous sequences and generates double-stranded breaks (DSBs) in target DNAs. This mechanism enables the host to defend against invading DNAs such as plasmids and viruses. However, whether such a CbAgo-mediated DNA cleavage is mutagenic remains unexplored. Here we demonstrate that CbAgo, directed by plasmid-encoded guide sequences, can cleave genome target sites and induce chromosome recombination between downstream homologous sequences in Escherichia coli. The recombination rate correlates well with pAgo DNA cleavage activity and the mechanistic study suggests the recombination involves DSBs and RecBCD processing. In RecA-deficient E. coli strain, guide-directed CbAgo cleavage on chromosomes severely impairs cell growth, which can be utilized as counter-selection to assist Lambda-Red recombineering. These findings demonstrate the guide-directed cleavage of pAgo on the host genome is mutagenic and can lead to different outcomes according to the function of the host DNA repair machinery. We anticipate this novel DNA-guided interference to be useful in broader genetic manipulation. Our study also provides an in vivo assay to characterize or engineer pAgo DNA cleavage activity.

Additional Information

The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. Version 1 - November 18, 2021; Version 2 - March 25, 2022; Version 3 - August 15, 2022. We thank Dr. Alexei A. Aravin, Dr. Andrey Kulbachinskiy, Dr. Daria Esyunina, and Dr. David R. F. Leach for helpful discussions. We thank Rochelle A. Diamond and Jamie Tijerina for their help in flow cytometry experiments. Author contributions: S.H. designed and carried out experiments, with K.W. and S.L.M. providing guidance. S.H., K.W., and S.L.M. wrote the manuscript. Funding: Caltech Rosen Bioengineering Center Award. Shurl and Kay Curci Foundation Award. Funding for open access charge: Caltech Rosen Bioengineering Center Award. Conflict of interest statement. None declared.

Attached Files

Submitted - 2021.11.17.469050v3.full.pdf

Supplemental Material - media-1.pdf


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August 20, 2023
January 18, 2024