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) has been pivotal in revealing their role in transcriptional regulation. However, most of the ligands described to date lack intra-G4 selectivity, severely limiting their potential for uncovering the biological function of individual G4s across the genome. To overcome these limitations, we developed ATENA (Approach to Target Exact Nucleic Acid alternative structures). ATENA relies on the chemical modification of established G4-ligands to enable their conjugation onto a catalytically inactive Cas9 protein (dCas9) using HaloTag, allowing for the targeting of individual G4s in living cells. We have systematically screened the length of the PEG-linkers connecting the G4-ligands to the HaloTag and sgRNA sequences to attain optimal G4 engagement both in vitro and in cells. Using optimized conditions, we leveraged ATENA to demonstrate how the selective targeting of the well-studied G4 in the promoter of the oncogene c-MYC suppresses its transcription exclusively from the P1 promoter. We also show that positioning ligands in the proximity of regulatory elements suppresses c-MYC transcription in a G4-independent manner, highlighting the importance of appropriate design to measure genuine G4-mediated transcriptional changes. We also demonstrate that selective targeting of a G4 in the PVT1 promoter can either stimulate or repress its transcription depending on the type of G4-ligand used, indicating that functional responses associated with G4-stabilization can highly depend on the type of ligand used. We further harnessed ATENA to study transcriptional perturbation associated with cell-specific G4s, revealing that the functional responses associated with these structures are tightly linked with the expression levels of the targeted gene. Our study provides critical insights into G4-based therapeutic design, offering an innovative platform to investigate G4 biology with high precision.