Nuclease dead Cas9 is a programmable roadblock for DNA replication

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 .