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

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81 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

Bibliographical note

Funding Information:
Meitian Wang, Vincent Olieric and Takashi Tomizaki at the Swiss Light Source (Paul Scherrer Institute, Villigen, Switzerland) for assistance with X-ray diffraction measurements; Netherlands Organization of Scientific Research (NWO) [ECHO grant 711013002 and NWO-TOP grant 714.015.001 to J.v.d.O.]; Swiss National Science Foundation (SNSF) Project Grant [SNSF 31003A 149393 to to M.J.]; European Molecular Biology Organization (EMBO) [ALTF 179-2015 and aALTF 509-2017 to to D.C.S.]; M.J. is International Research Scholar of the Howard Hughes Medical Institute and Vallee Scholar of the Bert L & NKug-gie Vallee Foundation; Vidi [864.14.002 to C.J.] of the Netherlands Organization for Scientific research. Conflict of interest statement. None declared.

Publisher Copyright:
© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.


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