RT Journal Article
SR Electronic
T1 Nuclease dead Cas9 is a programmable roadblock for DNA replication
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 455543
DO 10.1101/455543
A1 Whinn, Kelsey
A1 Kaur, Gurleen
A1 Lewis, Jacob S.
A1 Schauer, Grant
A1 Müller, Stefan
A1 Jergic, Slobodan
A1 Maynard, Hamish
A1 Yan Gan, Zhong
A1 Naganbabu, Matharishwan
A1 Bruchez, Marcel P.
A1 O’Donnell, Michael E.
A1 Dixon, Nicholas E.
A1 van Oijen, Antoine M.
A1 Ghodke, Harshad
YR 2018
UL http://biorxiv.org/content/early/2018/10/29/455543.1.abstract
AB 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.