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
The search for putative enzymes that can facilitate gene editing has recently focused its attention on Argonaute proteins from prokaryotes
We show our independent and contemporary work leading to the identification of a prokaryotic Argonaute (pAgo) from the strain CWBI 1009 of C. butyricum (CbcAgo thereafter), describing its properties in comparison to that of the CbAgo protein characterized in these articles, including a requirement for 5’-phosphorylated gDNA and a smaller minimum gDNA size required for full activity
Mass spectrometry of proteolytic Trypsin/Chemotrypsin digestion fragments of these recalcitrant contaminant proteins revealed them to be the GroEL chaperone and an N-terminal fragment of the Strep II-tagged CbcAgo proteins (Figure 1C). Presence of these co-purified proteins did not interfere with the capacity of the wild-type CbcAgo protein to cleave the ssDNA target after being preloaded with a complementary gDNA guide (Figure 2)
Summary
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. Methods: Here we search for and identify CbcAgo, a prokaryotic Argonaute protein from a mesophilic bacterium, and characterize in vitro its DNA interference activity. 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
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