G6PD CRISPR Activation Plasmid (m): sc-420446-ACT

Mouse G6pdx encodes glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the oxidative branch of the pentose phosphate pathway that converts glucose-6-phosphate to 6-phosphogluconolactone while generating NADPH. NADPH supports glutathione-dependent antioxidant defenses, redox homeostasis, and biosynthetic programs including lipid and nucleotide synthesis, thereby influencing proliferation and cellular stress responses. G6PD activity intersects with metabolic rewiring in immune activation and mitochondrial reactive oxygen species buffering, linking it to pathways that regulate inflammation, ferroptosis susceptibility, and proteostasis. Altered G6PD expression or activity is widely used as a model for studying oxidative stress–related phenotypes and metabolic vulnerabilities relevant to anemia-like redox defects and cardiometabolic and neuroinflammatory contexts.

G6PD CRISPR Activation Plasmid (m) provides a targeted, non-destructive approach to upregulating endogenous G6pdx expression without altering the underlying DNA sequence.

G6PD CRISPR Activation Plasmid (m) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the G6pdx locus in human cell lines. The system is built around a catalytically inactive Cas9 (dCas9) carrying two inactivating mutations (D10A and N863A) that eliminate nuclease activity while preserving DNA binding. This dCas9 is fused to VP64, a potent transcriptional activator, and is co-expressed with a blasticidin resistance gene for selection. The second plasmid encodes the MS2-p65-HSF1 fusion protein, a secondary activator complex that works in concert with dCas9-VP64, alongside a hygromycin resistance gene. The third plasmid encodes a target-specific 20 nt sgRNA fused to two MS2 RNA aptamers that recruit the MS2-p65-HSF1 complex to the activation site, accompanied by a puromycin resistance gene. The three plasmids are delivered at a 1:1:1 mass ratio for balanced expression of all system components.

Once assembled at the target locus, the SAM complex binds within approximately 200 bp upstream of the G6pdx transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous G6PD expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native G6pdx locus and enabling the study of G6PD-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of G6PD pathway restoration in tumor cells with silenced or reduced G6pdx expression.

For Research Use Only. Not Intended for Diagnostic or Therapeutic Use.