Versatile transcription control based on reversible dCas9 binding

The ability to control transcription in a time-dependent manner in vitro promises numerous applications in molecular biology and nanotechnology. Here we demonstrate an approach that enables precise, independent control over the production of multiple RNA transcripts in vitro using single guide RNA (sgRNA)-directed transcription blockades by catalytically dead Streptococcus pyogenes CRISPR-Cas9 enzyme (dCas9). We show that when bound to a DNA template, the dCas9:sgRNA complex forms a robust blockade to transcription by RNA polymerases (RNAPs) from bacteriophages SP6, T3, and T7 (>99.5% efficiency), and a partial blockade to transcription by Escherichia coli RNAP (∼70% efficiency). We find that all three bacteriophage RNAPs dissociate from the DNA template upon encountering the dCas9 blockade, while E. coli RNAP stays bound for at least the 90-min duration of our experiments. The blockade maintains >95% efficiency when four mismatches are introduced into the 5' end of the sgRNA target sequence. Notably, when using such a mismatched blockade, production of specific RNA species can be activated on demand by addition of a double-stranded competitor DNA perfectly matching the sgRNA. This strategy enables the independent production of multiple RNA species in a temporally controlled fashion from the same DNA template, demonstrating a new approach for transcription control.