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Published May 8, 2023 | Published + Supplemental Material
Journal Article Open

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 Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 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. DATA AVAILABILITY. Additional notes and data are available in the Supplemental materials. Conflict of interest statement. None declared.

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Published - gkad188.pdf

Supplemental Material - gkad188_supplemental_file.pdf


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August 22, 2023
January 18, 2024