G6PD CRISPR Activation Plasmid (h): sc-401019-ACT

Human G6PD encodes glucose-6-phosphate dehydrogenase, the rate-limiting enzyme of the oxidative pentose phosphate pathway that generates NADPH for reductive biosynthesis and antioxidant defense. By sustaining glutathione redox cycling and protecting against reactive oxygen species, G6PD supports cellular responses to oxidative and metabolic stress, particularly in erythrocytes and other high-oxidant environments. G6PD activity influences nucleotide biosynthesis via ribose-5-phosphate production and intersects with pathways governing proliferation, inflammation, and ferroptosis sensitivity. Genetic or functional perturbation of G6PD is associated with hemolytic susceptibility under oxidative challenge and is widely studied for its impact on redox homeostasis across diverse disease-relevant contexts.

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

G6PD CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the G6PD 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 G6PD 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 G6PD 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 G6PD expression.

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