GAD-67 CRISPR Activation Plasmid (h): sc-400588-ACT

GAD1 encodes glutamate decarboxylase 67 (GAD-67), a pyridoxal phosphate–dependent enzyme that catalyzes conversion of L-glutamate to γ-aminobutyric acid (GABA), supporting inhibitory neurotransmission and excitation–inhibition balance in the human central nervous system. GAD-67 contributes to cytosolic GABA pools in GABAergic interneurons and is linked to synaptic function, neuronal network oscillations, and activity-dependent metabolic coupling. Altered GAD1/GAD-67 expression has been associated with disrupted GABAergic signaling observed in multiple neuropsychiatric and neurodevelopmental conditions, making it a key target for mechanistic studies. Experimental modulation of GAD1 is commonly used to probe inhibitory circuit physiology, neurotransmitter homeostasis, and transcriptional regulation in neuronal models.

GAD-67 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous GAD1 expression without altering the underlying DNA sequence.

GAD-67 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the GAD1 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 GAD1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous GAD-67 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native GAD1 locus and enabling the study of GAD-67-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of GAD-67 pathway restoration in tumor cells with silenced or reduced GAD1 expression.

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