NNT CRISPR Activation Plasmid (h): sc-402908-ACT

Human NNT (nicotinamide nucleotide transhydrogenase) is an inner mitochondrial membrane enzyme that couples the proton motive force to the transfer of reducing equivalents between NAD(H) and NADP(H), sustaining mitochondrial NADPH pools. By supporting glutathione- and thioredoxin-dependent antioxidant systems, NNT helps control reactive oxygen species and preserves redox homeostasis during oxidative phosphorylation and metabolic stress. NNT activity influences mitochondrial respiration, steroidogenesis, and broader metabolic signaling through its impact on NADPH-dependent biosynthetic and detoxification pathways. Perturbation of NNT function has been linked to mitochondrial redox imbalance and has been studied in contexts including metabolic dysfunction and disorders associated with impaired oxidative stress buffering.

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

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

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