PT - JOURNAL ARTICLE AU - Sejla Salic AU - Anna Sieber AU - Samir Barbaria AU - Anatoly Vasilyev AU - Elisavet Papadimou AU - Nevena Milosavljevic AU - Benjamin Klapholz AU - Rohan Sivapalan AU - Trevor Collingwood AU - Tom Henley AU - Modassir Choudhry AU - Tilmann Bürckstümmer TI - A Bioinformatic and Empiric Exploration of Prokaryotic Argonautes as Novel Programmable Endonuclease Systems AID - 10.1101/2021.03.15.435463 DP - 2021 Jan 01 TA - bioRxiv PG - 2021.03.15.435463 4099 - http://biorxiv.org/content/early/2021/03/15/2021.03.15.435463.short 4100 - http://biorxiv.org/content/early/2021/03/15/2021.03.15.435463.full AB - Argonautes are nucleases that can be programmed by short oligonucleotides to cleave complementary sequences. Here, we performed an unbiased bioinformatic search to mine bacterial genomes for prokaryotic Argonautes (pAgos) harboring a PIWI domain. Our search identified 3,033 pAgos in total, of which 1,464 portend to the subgroup of long pAgos with more than 600 amino acids. We purified a subset of 49 pAgos which were found in proximity to helicases and tested their nuclease activity in vitro. Ten of these were active towards single-stranded DNA substrates and this activity could be programmed by exogenous guide DNAs or RNAs. Cleavage of double-stranded plasmid DNA was much less readily observed and was fostered by elevated temperatures or exogenous addition of a DNA single-strand binding protein (ET-SSB). The efficiency of pAgo-mediated plasmid cleavage was dependent on the DNA target sequence as well as the surrounding sequence, suggesting that unwinding of the DNA double helix was a limiting factor. Intriguingly, we identified a cluster of pAgos from the Clostridial clade which was active at 37°C and activity was enhanced by exogenous ET-SSB. This suggests that Clostridial pAgos may be particularly suited to catalyze DNA double-strand cleavage and implies that such pAgos may be repurposed as gene editing tools in future.Competing Interest StatementIntima Bioscience Inc has filed patent applications covering the intellectual property described within this manuscript