GLUD1 CRISPR Activation Plasmid (h): sc-402187-ACT

Human GLUD1 encodes mitochondrial glutamate dehydrogenase 1, an NAD(P)+-dependent enzyme that catalyzes the reversible oxidative deamination of glutamate to α-ketoglutarate and ammonia, linking amino acid catabolism to the TCA cycle. By regulating the balance between glutamate utilization and anaplerotic flux, GLUD1 influences cellular bioenergetics, redox homeostasis, and nitrogen metabolism, with downstream effects on neurotransmitter pools and metabolic coupling. GLUD1 activity interfaces with pathways controlling mitochondrial function and metabolic signaling, and altered regulation has been associated with disorders involving amino acid handling and neuroendocrine metabolic dysregulation. These features make GLUD1 a relevant target for mechanistic studies of mitochondrial metabolism and glutamate-dependent cellular phenotypes.

GLUD1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous GLUD1 expression without altering the underlying DNA sequence.

GLUD1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the GLUD1 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 GLUD1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous GLUD1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native GLUD1 locus and enabling the study of GLUD1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of GLUD1 pathway restoration in tumor cells with silenced or reduced GLUD1 expression.

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