Glyoxalase I CRISPR Activation Plasmid (h): sc-401914-ACT

Human GLO1 encodes glyoxalase I, a zinc-dependent metalloenzyme that catalyzes the first and rate-limiting step of the glyoxalase system, converting the cytotoxic glycolytic byproduct methylglyoxal (as a hemithioacetal with glutathione) into S‑D‑lactoylglutathione. By controlling methylglyoxal levels, GLO1 helps limit non-enzymatic protein and nucleic acid glycation and reduces formation of advanced glycation end products, thereby influencing redox homeostasis and cellular stress responses. GLO1 activity is linked to metabolic rewiring and detoxification capacity, processes frequently interrogated in models of oxidative stress, inflammation, and altered glucose metabolism. Dysregulated methylglyoxal clearance and glycation burden are relevant to disease-associated phenotypes including insulin resistance, vascular dysfunction, and tumor cell stress tolerance, supporting the use of GLO1 as a functional node in metabolism and proteostasis research.

Glyoxalase I CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous GLO1 expression without altering the underlying DNA sequence.

Glyoxalase I CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the GLO1 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 GLO1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Glyoxalase I expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native GLO1 locus and enabling the study of Glyoxalase I-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Glyoxalase I pathway restoration in tumor cells with silenced or reduced GLO1 expression.

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