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DNA-guided DNA cleavage at moderate temperatures by Clostridium butyricum Argonaute

By Jorrit W. Hegge, Daan C. Swarts, Stanley D. Chandradoss, Tao Ju Cui, Jeroen Kneppers, Martin Jinek, Chirlmin Joo, John van der Oost

Posted 29 Jan 2019
bioRxiv DOI: 10.1101/534206 (published DOI: 10.1093/nar/gkz306)

Prokaryotic Argonaute proteins (pAgos) constitute a diverse group of endonucleases of which some mediate host defense by utilizing small interfering DNA guides (siDNA) to cleave complementary invading DNA. This activity can been repurposed for programmable DNA cleavage. However, currently characterized DNA-cleaving pAgos require elevated temperatures (>65°C) for their activity, making them less suitable for applications that require moderate temperatures, such as genome editing. Here we report the functional and structural characterization of the pAgo from the mesophilic bacterium Clostridium butyricum (CbAgo). CbAgo can be reprogrammed with siDNAs to cleave complementary DNA, but not RNA. CbAgo displays a preference for siDNAs that have a deoxyadenosine at the 5′-end and thymidines in the sub-seed segment (siDNA nucleotides 2-4). Furthermore, CbAgo mediates DNA-guided DNA cleavage of AT-rich double stranded DNA at moderate temperatures (37°C). This study demonstrates that certain pAgos are capable of programmable DNA cleavage at moderate temperatures and thereby expands the scope of the potential pAgo-based applications.

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