Transcription Regulation by Targeting G-quadruplex Forming Sequences with CRISPR/dCas9*

Nuclease-dead Cas9 (dCas9) enables transient and sequence specific transcription regelation when putative quadruplex forming sequences (PQS) are targeted. We present functional in cellulo experiments where we monitored mRNA and protein expression levels and in vitro bulk and single molecule experiments where we investigated how RNA polymerase (RNAP) interacts with dCas9 and G-quadruplex (G4 or GQ) structures. We used the PQS within the human tyrosine hydroxylase (TH) and c-Myc promoters as model systems as they can form multiple G4 structures with different stabilities. In both systems, we demonstrate that transcription levels can be up or down regulated by targeting different parts of these G-rich sequences with dCas9. We demonstrate that dCas9 targeting, which is specific, transient and does not result in sequence modifications, yields similar levels of control to those attained with site directed mutagenesis and small molecule studies. To gain mechanistic understanding of these observations, we performed in vitro RNAP stop assay and single molecule FRET assays where the location and orientation of PQS was varied with respect to T7 RNAP promoter site and dCas9 target site. These studies shed light on the complex interactions between G4, CRISPR/dCas9, and RNAP and highlight the potent and versatile capabilities of dCas9 in gene expression regulation.