CRISPR-Cas9 Targeting of G-Quadruplex DNA in ADH1 promoter Highlights its role in Transcriptome and Metabolome Regulation

G-quadruplex (G4) structures are critical regulators of gene expression, yet the role of an individual G4 within its native chromatin remains underexplored. Here, we used CRISPR-Cas9 to introduce guanine-to-thymine mutations at a G4-forming motif within the adh1 ⁺ promoter in yeast, creating two mutant strains: one with G4-only mutations and another with both G4 and TATA-box mutations. Chromatin immunoprecipitation using BG4 antibody confirmed reduced G4 enrichment in both mutants, validating G4 structure formation in the wild-type chromatin. Detailed characterizations demonstrated that the G4 mutations alter its dynamics without fully preventing its formation. These mutations significantly reduce adh1 transcript levels, with G4 TATA-box mutant causing the strongest transcriptional suppression. This indicates a positive regulatory role for the Adh1 G4 structure in adh1 ⁺ gene expression. Furthermore, both mutants displayed altered transcriptomic profiles, particularly impacting the oxidoreductase pathway. Metabolomic analyses by mass spectrometry further highlighted substantial disruptions in NAD+/NADH metabolism, a key energy reservoir for metabolic regulation. Together, our findings illustrate how deregulation of a single G4 structure influences transcriptome regulation, with implications for metabolic diseases. It also highlights the therapeutic potential of G4 modulation as a novel, controlled approach to reprogram cellular metabolism to achieve targeted phenotypic shifts.