GSS CRISPR Activation Plasmid (h): sc-402992-ACT

Human GSS encodes glutathione synthetase, the ATP-dependent enzyme that catalyzes the final step of glutathione (GSH) biosynthesis by ligating glycine to γ-glutamylcysteine. By controlling intracellular GSH pools, GSS is central to redox homeostasis, detoxification of electrophiles via glutathione S-transferase pathways, and protection against oxidative damage that influences mitochondrial function and cell survival signaling. GSS activity shapes cellular responses to reactive oxygen species and supports metabolic adaptation during stress, linking it to processes such as ferroptosis susceptibility, xenobiotic metabolism, and inflammatory signaling. Genetic or functional impairment of GSS is associated with glutathione deficiency and oxidative stress phenotypes, providing a mechanistic connection to hematologic and neurologic dysfunction and broader redox-related disease biology.

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

GSS CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the GSS 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 GSS transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous GSS expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native GSS locus and enabling the study of GSS-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of GSS pathway restoration in tumor cells with silenced or reduced GSS expression.

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