OPLAH CRISPR Activation Plasmid (h): sc-411939-ACT

OPLAH encodes 5-oxoprolinase, a cytosolic enzyme that converts 5-oxoproline (pyroglutamate) to L-glutamate in an ATP-dependent step of the γ-glutamyl cycle. Through this reaction, OPLAH contributes to glutathione turnover, cellular redox homeostasis, and amino acid metabolism, linking oxidative stress responses with mitochondrial and cytosolic metabolic flux. Altered OPLAH activity can perturb 5-oxoproline handling and glutamate availability, processes relevant to metabolic stress phenotypes and redox-imbalanced states. Because the γ-glutamyl cycle intersects with detoxification pathways and antioxidant capacity, OPLAH regulation is of interest in studies of oxidative injury, inflammation-associated metabolism, and neurometabolic signaling.

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

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

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