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

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

Research output: Contribution to journalArticlepeer-review

57 Scopus citations


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 be repurposed for programmable DNA cleavage. However, currently characterized DNA-cleaving pAgos require elevated temperatures (≥65C) 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 siDNA-guided DNA-targeting pAgo from the mesophilic bacterium Clostridium butyricum (CbAgo). CbAgo displays a preference for siDNAs that have a deoxyadenosine at the 5'-end and thymidines at nucleotides 2-4. Furthermore, CbAgo mediates DNA-guided DNA cleavage of AT-rich double stranded DNA at moderate temperatures (37C). 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.

Original languageEnglish
Pages (from-to)5809-5821
Number of pages13
JournalNucleic Acids Research
Issue number11
StatePublished - 20 Jun 2019


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