Prokaryotic Argonautes (pAgos) are programmable nucleases involved in cell defense against invading DNA. In vitro, pAgos can bind small single-stranded guide DNAs to recognize and cleave complementary DNA. In vivo, pAgos preferentially target plasmids, phages and multicopy genetic elements. Here, we show that CbAgo nuclease from Clostridium butyricum can be used for genomic DNA engineering in bacteria. We demonstrate that CbAgo loaded with plasmid-derived guide DNAs can recognize and cleave homologous chromosomal loci, and define the minimal length of homology required for this targeting. Cleavage of plasmid DNA at an engineered site of the I-SceI meganuclease increases guide DNA loading into CbAgo and enhances processing of homologous chromosomal loci. Analysis of guide DNA loading into CbAgo also reveals off-target sites of I-SceI in the Escherichia coli genome, demonstrating that pAgos can be used for highly sensitive detection of double-stranded breaks in genomic DNA. Finally, we show that CbAgo-dependent targeting of genomic loci with plasmid-derived guide DNAs promotes homologous recombination between plasmid and chromosomal DNA, depending on the catalytic activity of CbAgo. Specific targeting of plasmids with Argonautes can be used to integrate plasmid-encoded sequences into the chromosome thus enabling genome editing.

Prokaryotic Argonautes (pAgos) are programmable nucleases involved in cell defense against invading DNA. Recent studies showed that pAgos can bind small single-stranded guide DNAs (gDNAs) to recognize and cleave complementary DNA in vitro. In vivo pAgos preferentially target plasmids, phages and multicopy genetic elements. Here, we reveal that CbAgo nuclease from Clostridium butyricum can be used for genomic DNA cleavage and engineering in bacteria. CbAgo-dependent targeting of genomic loci with plasmid-derived gDNAs promotes recombination between plasmid and chromosomal DNA. Efficient genome cleavage and recombineering depends on the catalytic activity of CbAgo, its interactions with gDNAs, and the extent of homology between plasmid and chromosomal sequences. Specific targeting of plasmids with Argonautes can be used to integrate plasmid-encoded sequences into the chromosome thus enabling genome editing.