Chemically modified CRISPR-Cas9 enables targeting of individual G-quadruplex and i-motif structures, revealing ligand-dependent transcriptional perturbation

The development of selective ligands to target DNA G-quadruplexes (G4s) and i-motifs (iMs) has revealed their relevance in transcriptional regulation. However, most of these ligands are unable to target individual G4s or iMs in the genome, limiting their scope. Herein, we describe an Approach to Target Exact Nucleic Acid alternative structures (ATENA) that relies on the chemical conjugation of established G4 and iM ligands to a catalytically inactive Cas9 protein (dCas9), enabling their individual targeting in living cells. ATENA demonstrates that the selective targeting of the G4 present in the oncogene c-MYC leads to the suppression of transcripts regulated exclusively by one of its promoters (P1). Conversely, targeting the c-MYC iMs on the opposite strand leads to the selective increase of P1-driven transcripts. ATENA reveals that G4-mediated transcriptional responses are highly ligand-specific, with different ligands eliciting markedly different effects at the same G4 site. We further demonstrate that the basal expression levels of the gene targeted can be used to predict the transcriptional impact associated with G4-stabilization. Our study provides a platform for investigating G4- and iM-biology with high precision, unveiling the therapeutic relevance of individual DNA structures with selectivity.