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Nuclease dead Cas9 is a programmable roadblock for DNA replication

Kelsey Whinn, Gurleen Kaur, Jacob S. Lewis, Grant Schauer, Stefan Müller, Slobodan Jergic, Hamish Maynard, Zhong Yan Gan, Matharishwan Naganbabu, Marcel P. Bruchez, Michael E. O’Donnell, Nicholas E. Dixon, Antoine M. van Oijen, Harshad Ghodke
doi: https://doi.org/10.1101/455543
Kelsey Whinn
1School of Chemistry and Molecular Bioscience and Molecular Horizons, University of Wollongong, Wollongong, New South Wales 2522, Australia
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Gurleen Kaur
1School of Chemistry and Molecular Bioscience and Molecular Horizons, University of Wollongong, Wollongong, New South Wales 2522, Australia
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Jacob S. Lewis
1School of Chemistry and Molecular Bioscience and Molecular Horizons, University of Wollongong, Wollongong, New South Wales 2522, Australia
2Illawarra Health and Medical Research Institute, Wollongong, New South Wales 2522, Australia
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Grant Schauer
3Howard Hughes Medical Institute, Rockefeller University, New York, NY 10065, USA
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Stefan Müller
1School of Chemistry and Molecular Bioscience and Molecular Horizons, University of Wollongong, Wollongong, New South Wales 2522, Australia
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Slobodan Jergic
1School of Chemistry and Molecular Bioscience and Molecular Horizons, University of Wollongong, Wollongong, New South Wales 2522, Australia
2Illawarra Health and Medical Research Institute, Wollongong, New South Wales 2522, Australia
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Hamish Maynard
1School of Chemistry and Molecular Bioscience and Molecular Horizons, University of Wollongong, Wollongong, New South Wales 2522, Australia
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Zhong Yan Gan
1School of Chemistry and Molecular Bioscience and Molecular Horizons, University of Wollongong, Wollongong, New South Wales 2522, Australia
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Matharishwan Naganbabu
4Department of Chemistry and Center for Nucleic Acids Science and Technology, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213, USA
5Department of Biological Sciences and Molecular Biosensor and Imaging Center, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213, USA
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Marcel P. Bruchez
4Department of Chemistry and Center for Nucleic Acids Science and Technology, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213, USA
5Department of Biological Sciences and Molecular Biosensor and Imaging Center, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213, USA
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Michael E. O’Donnell
3Howard Hughes Medical Institute, Rockefeller University, New York, NY 10065, USA
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Nicholas E. Dixon
1School of Chemistry and Molecular Bioscience and Molecular Horizons, University of Wollongong, Wollongong, New South Wales 2522, Australia
2Illawarra Health and Medical Research Institute, Wollongong, New South Wales 2522, Australia
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Antoine M. van Oijen
1School of Chemistry and Molecular Bioscience and Molecular Horizons, University of Wollongong, Wollongong, New South Wales 2522, Australia
2Illawarra Health and Medical Research Institute, Wollongong, New South Wales 2522, Australia
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Harshad Ghodke
1School of Chemistry and Molecular Bioscience and Molecular Horizons, University of Wollongong, Wollongong, New South Wales 2522, Australia
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Abstract

DNA replication occurs on chromosomal DNA while processes such as DNA repair, recombination and transcription continue. However, we have limited experimental tools to study the consequences of collisions between DNA-bound molecular machines. Here, we repurpose a catalytically inactivated Cas9 (dCas9) construct fused to the photo-stable dL5 protein fluoromodule as a novel, targetable protein-DNA roadblock for studying replication fork arrest at the single-molecule level in vitro as well as in vivo. We find that the specifically bound dCas9–guideRNA complex arrests viral, bacterial and eukaryotic replication forks in vitro.

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Posted October 29, 2018.
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Nuclease dead Cas9 is a programmable roadblock for DNA replication
Kelsey Whinn, Gurleen Kaur, Jacob S. Lewis, Grant Schauer, Stefan Müller, Slobodan Jergic, Hamish Maynard, Zhong Yan Gan, Matharishwan Naganbabu, Marcel P. Bruchez, Michael E. O’Donnell, Nicholas E. Dixon, Antoine M. van Oijen, Harshad Ghodke
bioRxiv 455543; doi: https://doi.org/10.1101/455543
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Nuclease dead Cas9 is a programmable roadblock for DNA replication
Kelsey Whinn, Gurleen Kaur, Jacob S. Lewis, Grant Schauer, Stefan Müller, Slobodan Jergic, Hamish Maynard, Zhong Yan Gan, Matharishwan Naganbabu, Marcel P. Bruchez, Michael E. O’Donnell, Nicholas E. Dixon, Antoine M. van Oijen, Harshad Ghodke
bioRxiv 455543; doi: https://doi.org/10.1101/455543

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