A step by step protocol for stabilizing specific DNA G-quadruplexes by CRISPR guided G-quadruplex binding proteins and ligands

Abstract Despite the importance of DNA G-quadruplexes (G4s) in health and disease being demonstrated, technologies to readily manipulate specific G4 folding for functional analysis and therapeutic purposes are lacking. Here, we employ the G4-stabilizing protein/ligand in conjunction with CRISPR system to selectively facilitate single or multiple targeted G4 folding within specific genomic loci. We demonstrate that fusion of nucleolin with a catalytically inactive Cas9 (dCas9) can specifically stabilize G4s in the promoter of oncogene MYC and muscle associated gene Itga7 as well as telomere G4s, leading to cell proliferation arrest, inhibition of myoblast differentiation and cell senescence, respectively. Furthermore, CRISPR system can confer intra-G4 selectivity to G4-binding compounds PDC and PDS. Compared with traditional G4 ligands, CRISPR-guided Bio-PDC enables a more precise investigation into the biological functionality of de novo G4s. Our study provides insights that will enhance understanding of G4 functions and therapeutic interventions.