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DOI

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

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.

Original languageEnglish
Pages (from-to)5809-5821
JournalNucleic Acids Research
Volume47
Issue number11
DOIs
Publication statusPublished - 2019

ID: 55178412