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, especially outside human systems. Here, we used CRISPR-Cas9 to introduce guanine-to-thymine mutations at a G4-forming motif within the adh1+ promoter in yeast Schizosaccharomyces pombe, 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 G4 structure in transcription. 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. These results highlight that tuning G4 dynamics, without abolishing the structure, can still profoundly affect gene expression and metabolism, unlike prior studies on the human MYC promoter that disrupted G4 formation. This represents the first such finding in yeast.