Abstract Argonaute proteins are an evolutionarily conserved family of proteins capable of recognizing and cleaving specific nucleic acid sequences using complementary guide molecules. Eukaryotic Argonautes play a key role in RNA interference by utilizing short RNAs of various classes to recognize target mRNAs. Prokaryotic Argonautes are much more diverse and most of them recognize DNA targets. The search for new Argonautes that would be active under varying conditions is important for both understanding their functions and developing new tools for genetic technologies. Many previously studied Argonautes exhibit low activity at low and moderate temperatures. To overcome this limitation, we isolated and studied two Argonaute proteins from psychrotolerant cyanobacteria, CstAgo from Cyanobacterium stanieri and CspAgo from Calothrix sp . Both proteins use short DNA guides to recognize and cleave DNA targets. CstAgo displayed no specificity for the 5′-end structure of the guide, while CspAgo de
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