The Argonaute (Ago) enzyme complex plays a critical role in DNA and RNA target cleavage for a process known as RNA silencing in prokaryotic and eukaryotic cells, making them a target for future gene-editing technology. The present study unravels key differences between prokaryotic Ago (pAgo) and eukaryotic Ago (eAgo) enzymes in the cleavage reaction and may provide important clues on their evolutionary past.