Nuclease dead Cas9 is a programmable roadblock for DNA replication

Kelsey S Whinn; Gurleen Kaur; Jacob S Lewis; Grant D Schauer; Stefan H Mueller; 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:10.1038/s41598-019-49837-z

Nuclease dead Cas9 is a programmable roadblock for DNA replication

Sci Rep. 2019 Sep 16;9(1):13292. doi: 10.1038/s41598-019-49837-z.

Authors

Kelsey S Whinn^{1

2}, Gurleen Kaur^{1

2}, Jacob S Lewis^{1

2}, Grant D Schauer³, Stefan H Mueller^{1

2}, Slobodan Jergic^{1

2}, Hamish Maynard¹, Zhong Yan Gan¹, Matharishwan Naganbabu⁴, Marcel P Bruchez^{4

5}, Michael E O'Donnell³, Nicholas E Dixon^{1

2}, Antoine M van Oijen^{6

7}, Harshad Ghodke^{8

9}

Affiliations

¹ School of Chemistry and Molecular Bioscience and Molecular Horizons, University of Wollongong, Wollongong, New South Wales, 2522, Australia.
² Illawarra Health and Medical Research Institute, Wollongong, New South Wales, 2522, Australia.
³ Howard Hughes Medical Institute, Rockefeller University, New York, NY, 10065, USA.
⁴ Department of Chemistry and Molecular Biosensors and Imaging Center, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, Pennsylvania, 15213, USA.
⁵ Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, Pennsylvania, 15213, USA.
⁶ School of Chemistry and Molecular Bioscience and Molecular Horizons, University of Wollongong, Wollongong, New South Wales, 2522, Australia. vanoijen@uow.edu.au.
⁷ Illawarra Health and Medical Research Institute, Wollongong, New South Wales, 2522, Australia. vanoijen@uow.edu.au.
⁸ School of Chemistry and Molecular Bioscience and Molecular Horizons, University of Wollongong, Wollongong, New South Wales, 2522, Australia. harshad@uow.edu.au.
⁹ Illawarra Health and Medical Research Institute, Wollongong, New South Wales, 2522, Australia. harshad@uow.edu.au.

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.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

CRISPR-Associated Protein 9 / genetics*
CRISPR-Cas Systems / genetics
DNA Replication / genetics*
DNA, Bacterial / genetics*
Escherichia coli / genetics*
RNA, Guide, CRISPR-Cas Systems
Streptococcus pyogenes / enzymology

Substances

DNA, Bacterial
CRISPR-Associated Protein 9

Abstract

Publication types

MeSH terms

Substances

Grants and funding