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Nuclease dead Cas9 is a programmable roadblock for DNA replication.

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.

SUBMITTER: Whinn KS

PROVIDER: S-EPMC6746809 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Nuclease dead Cas9 is a programmable roadblock for DNA replication.

Whinn Kelsey S KS Kaur Gurleen G Lewis Jacob S JS Schauer Grant D GD Mueller Stefan H SH Jergic Slobodan S Maynard Hamish H Gan Zhong Yan ZY Naganbabu Matharishwan M Bruchez Marcel P MP O'Donnell Michael E ME Dixon Nicholas E NE van Oijen Antoine M AM Ghodke Harshad H

Scientific reports 20190916 1

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 eukaryo ...[more]

PMID: 31527759

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