Nuclease dead Cas9 is a programmable roadblock for DNA replication

Authors

Kelsey Whinn, University of WollongongFollow
Gurleen Kaur, University of WollongongFollow
Jacob S. Lewis, University of WollongongFollow
Grant D. Schauer, Rockefeller University, Carnegie Mellon University, University of Pittsburgh
Stefan Mueller, University of WollongongFollow
Slobodan Jergic, University of WollongongFollow
Hamish Maynard, University of Wollongong
Zhong Gan, University of Wollongong
Matharishwan Naganbabu, Carnegie Mellon University
Marcel Bruchez, Carnegie Mellon University
Michael E. O'Donnell, Rockefeller University
Nicholas E. Dixon, University of WollongongFollow
Antoine M. van Oijen, University of WollongongFollow
Harshad Ghodke, University of WollongongFollow

RIS ID

139027

Publication Details

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.

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.

Grant Number

ARC/DP180100858

Grant Number

ARC/FL140100027