Nuclease dead Cas9 is a programmable roadblock for DNA replication

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 .