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Nuclease dead Cas9 is a programmable roadblock for DNA replication

Journal Article


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Abstract


  • Limited experimental tools are available to study the consequences of collisions between DNA-bound molecular machines. Here, we repurpose a catalytically inactivated Cas9 (dCas9) construct as a generic, novel, targetable protein–DNA roadblock for studying mechanisms underlying enzymatic activities on DNA substrates in vitro. We illustrate the broad utility of this tool by demonstrating replication fork arrest by the specifically bound dCas9–guideRNA complex to arrest viral, bacterial and eukaryotic replication forks in vitro.

Authors


  •   Whinn, Kelsey (external author)
  •   Kaur, Gurleen (external author)
  •   Lewis, Jacob S. (external author)
  •   Schauer, Grant (external author)
  •   Mueller, Stefan (external author)
  •   Jergic, Slobodan
  •   Maynard, Hamish (external author)
  •   Gan, Zhong (external author)
  •   Naganbabu, Matharishwan (external author)
  •   Bruchez, Marcel (external author)
  •   O'Donnell, Michael E. (external author)
  •   Dixon, Nicholas E.
  •   van Oijen, Antoine M.
  •   Ghodke, Harshad

Publication Date


  • 2019

Citation


  • Whinn, K., Kaur, G., Lewis, J. S., Schauer, G. D., Mueller, S. H., Jergic, S., Maynard, H., Gan, Z. Yan., Naganbabu, M., Bruchez, M. P., O'Donnell, M. E., Dixon, N. E., van Oijen, A. M. & Ghodke, H. (2019). Nuclease dead Cas9 is a programmable roadblock for DNA replication. Scientific Reports, 9 13292-1-13292-9.

Ro Full-text Url


  • https://ro.uow.edu.au/cgi/viewcontent.cgi?article=1942&context=smhpapers1

Ro Metadata Url


  • http://ro.uow.edu.au/smhpapers1/928

Start Page


  • 13292-1

End Page


  • 13292-9

Volume


  • 9

Place Of Publication


  • United Kingdom

Abstract


  • Limited experimental tools are available to study the consequences of collisions between DNA-bound molecular machines. Here, we repurpose a catalytically inactivated Cas9 (dCas9) construct as a generic, novel, targetable protein–DNA roadblock for studying mechanisms underlying enzymatic activities on DNA substrates in vitro. We illustrate the broad utility of this tool by demonstrating replication fork arrest by the specifically bound dCas9–guideRNA complex to arrest viral, bacterial and eukaryotic replication forks in vitro.

Authors


  •   Whinn, Kelsey (external author)
  •   Kaur, Gurleen (external author)
  •   Lewis, Jacob S. (external author)
  •   Schauer, Grant (external author)
  •   Mueller, Stefan (external author)
  •   Jergic, Slobodan
  •   Maynard, Hamish (external author)
  •   Gan, Zhong (external author)
  •   Naganbabu, Matharishwan (external author)
  •   Bruchez, Marcel (external author)
  •   O'Donnell, Michael E. (external author)
  •   Dixon, Nicholas E.
  •   van Oijen, Antoine M.
  •   Ghodke, Harshad

Publication Date


  • 2019

Citation


  • Whinn, K., Kaur, G., Lewis, J. S., Schauer, G. D., Mueller, S. H., Jergic, S., Maynard, H., Gan, Z. Yan., Naganbabu, M., Bruchez, M. P., O'Donnell, M. E., Dixon, N. E., van Oijen, A. M. & Ghodke, H. (2019). Nuclease dead Cas9 is a programmable roadblock for DNA replication. Scientific Reports, 9 13292-1-13292-9.

Ro Full-text Url


  • https://ro.uow.edu.au/cgi/viewcontent.cgi?article=1942&context=smhpapers1

Ro Metadata Url


  • http://ro.uow.edu.au/smhpapers1/928

Start Page


  • 13292-1

End Page


  • 13292-9

Volume


  • 9

Place Of Publication


  • United Kingdom