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

• Published in: Nucleic acids research Vol. 47; no. 11; pp. 5809 - 5821
• Main Authors: Hegge, Jorrit W, Swarts, Daan C, Chandradoss, Stanley D, Cui, Tao Ju, Kneppers, Jeroen, Jinek, Martin, Joo, Chirlmin, van der Oost, John
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 20.06.2019
• Subjects: Argonaute Proteins - genetics; Argonaute Proteins - metabolism; BacGen; Bacterial Proteins - metabolism; Biochemie; Biochemistry; Clostridium butyricum - genetics; Clostridium butyricum - metabolism; DNA - chemistry; DNA - metabolism; DNA Cleavage; DNA, Single-Stranded - analysis; Fluorescence Resonance Energy Transfer; Gene Editing; Gene Silencing; Molecular Biology; Mutation; Phylogeny; Plasmids - metabolism; Protein Binding; RNA, Guide, CRISPR-Cas Systems; Temperature; VLAG
• ISSN: 0305-1048; 1362-4962
• 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 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.