BPS2026 – Harnessing G-quadruplexes for programmable control of transcription and translation

We present our recent investigations using nuclease-dead CRISPR-dCas9 and CRISPR-dCas13d to regulate c-Myc transcription and VEGF translation, respectively, by targeting transcriptionally or translationally active G-quadruplex (GQ or G4) structures. We quantified the mRNA and protein levels in Burkitt’s lymphoma cells ( c-Myc ) and human umbilical vein endothelial cells ( VEGF ) before and after targeting relevant regions. In addition, we conducted in vitro bulk and single-molecule experiments to probe the underlying regulatory mechanisms. Our results show that c-Myc transcription can be both up- and downregulated by combining CRISPR interference and activation with dCas9, while VEGF expression can be suppressed by dCas13d under hypoxic conditions. A variety of methods—including RT-qPCR, western blotting, cell viability assays, fluorescence beacon assays, and single-molecule techniques (smFRET and mass photometry)—were employed throughout these studies. This approach provides sequence-specific and transient gene regulation, avoiding the limitations of G4-stabilizing small molecules, which lack sequence specificity, and site-directed mutations, which are permanent. Furthermore, we demonstrate that the regulatory range can be significantly expanded when multiple inhibitory sites are targeted with dCas9 simultaneously. Overall, our study introduces a versatile, sequence-specific, and transient technology for regulating gene expression, with broad applicability to other genes containing transcriptionally or translationally active secondary structures.