Oct 29, 2018

Nuclease dead Cas9 is a programmable roadblock for DNA replication

BioRxiv : the Preprint Server for Biology
Kelsey WhinnHarshad Ghodke

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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Mentioned in this Paper

In Vivo
DNA Repair
Transcription, Genetic
Virus Replication
Recombination, Genetic
Collision
Replication Fork Arrest
Nuclease
Chromosomes
DNA Replication

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