Programmable Dna Cleavage by Cyanobacterial Argonautes

Argonautes are an evolutionarily conserved family of proteins that can recognize and cleave nucleic acids using complementary guide molecules. Eukaryotic Argonautes play a major role in RNA interference, using small RNAs to target mRNA. Prokaryotic Argonautes are much more diverse and predominantly recognize target DNA. Studying new Argonaute proteins, which are active under a wide range of conditions, is important for both understanding their functions and developing new genetic tools. The disadvantage of many previously studied Argonautes is their low activity at low and moderate temperatures. In this work, we isolated and characterized Argonautes from psychrotolerant cyanobacteria and ., CstAgo and CspAgo. Both proteins use short DNA guides to recognize DNA targets. CstAgo does not have strong preferences for the structure of the 5' end of guide DNA, while CspAgo demonstrates a weak preference for the 5'-terminal nucleotide. CstAgo has higher activity and is able to cleave single-stranded DNA at temperatures between 10 and 50 °C. CspAgo is more cold-sensitive but is capable of cleaving double-stranded plasmid DNA using specific guides. Therefore, the studied proteins could potentially be used for genetic manipulations of DNA.