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Nucleic Acids Res. 2019 May 9. pii: gkz306. doi: 10.1093/nar/gkz306. [Epub ahead of print]

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

Author information

1
Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, 6708 WE Wageningen, The Netherlands.
2
Laboratory of Biochemistry, Department of Agrotechnology and Food Sciences, Wageningen University, 6708 WE Wageningen, The Netherlands.
3
Department of Biochemistry, University of Zurich, CH-8057 Zurich, Switzerland.
4
Kavli Institute of NanoScience, Department of BioNanoScience, Delft University of Technology, 2629 HZ Delft, The Netherlands.

Abstract

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 (≥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 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 (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.

PMID:
31069393
DOI:
10.1093/nar/gkz306

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