Abstract
Background: The search for putative enzymes that can facilitate gene editing has recently focused its attention on Argonaute proteins from prokaryotes (pAgos). Though they are structural homologues of human Argonaute protein, which uses RNA guides to interfere with RNA targets, pAgos use ssDNA guides to identify and, in many cases, cut a complementary DNA target. Thermophilic pAgos from Thermus thermophilus, Pyrococcus furiosus and Methanocaldococcus jasmanii have been identified and thoroughly studied, but their thermoactivity makes them of little use in mesophilic systems such as mammalian cells. Methods: Here we search for and identify CbcAgo, a prokaryotic Argonaute protein from a mesophilic bacterium, and characterize in vitro its DNA interference activity. Results: CbcAgo efficiently uses 5'P-ssDNA guides as small as 11-mers to cut ssDNA targets, requires divalent cations (preferentially, Mn 2+) and has a maximum activity between 37 and 42 °C, remaining active up to 55 °C. Nicking activity on supercoiled dsDNA was shown. However, no efficient double-strand breaking activity could be demonstrated. Conclusions: CbcAgo can use gDNA guides as small as 11 nucleotides long to cut complementary ssDNA targets at 37ºC, making it a promising starting point for the development of new gene editing tools for mammalian cells.
Highlights
Argonaute proteins play a central role in gene silencing and defense against external RNA in eukaryotes, binding small RNA molecules that are used as guides to scan for complementary RNA targets in the form of mRNAs or RNA viruses
Homologues to eukaryotic Argonaute proteins (eAgo) that contain these four domains are found in both bacteria and archaea, being collectively known as prokaryotic Argonaute proteins
We show our independent work leading to the identification of a similar prokaryotic Argonaute (pAgo) (CbcAgo) in the strain CWBI 1009 of C. butyricum, describing its properties in comparison to that of the CbAgo protein described in the preprint article, including a higher maximum temperature of activity, a strict requirement for 5’-phosphorylated gDNA and a smaller minimum gDNA size required for full activity
Summary
Argonaute proteins play a central role in gene silencing and defense against external RNA in eukaryotes, binding small RNA molecules that are used as guides to scan for complementary RNA targets in the form of mRNAs or RNA viruses. Homologues to eAgo that contain these four domains are found in both bacteria and archaea, being collectively known as prokaryotic Argonaute (pAgo) proteins. Their function seems to depend on the species, targeting either RNA or DNA2. PAgo from Aquifex aeolicus (AeAgo)[6] and Natronobacterium gregoryi (NgAgo)[7] use ssDNA guides to target RNA, suggesting a putative role in gene silencing, similar to that of eAgo, or in defense against RNA viruses. Other pAgos, like that of Rhodobacter spheroides (RsAgo), use RNA guides against DNA targets, maintaining its defense capability against invading DNA despite the absence of endonucleolytic activity in its PIWI domain[8]. We show our independent work leading to the identification of a similar pAgo (CbcAgo) in the strain CWBI 1009 of C. butyricum, describing its properties in comparison to that of the CbAgo protein described in the preprint article, including a higher maximum temperature of activity, a strict requirement for 5’-phosphorylated gDNA and a smaller minimum gDNA size required for full activity
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