GAD-65 CRISPR Activation Plasmid (h): sc-402137-ACT

GAD2 encodes glutamate decarboxylase 65 (GAD-65), a pyridoxal phosphate–dependent enzyme that catalyzes the decarboxylation of glutamate to γ-aminobutyric acid (GABA), a principal inhibitory neurotransmitter in the human central nervous system. GAD-65 is enriched at presynaptic terminals where it supports activity-dependent GABA synthesis and contributes to synaptic vesicle cycling and inhibitory circuit homeostasis. By regulating the balance between excitation and inhibition, GAD2 influences neuronal signaling networks involved in neurodevelopment, synaptic plasticity, and stress-responsive circuitry. Altered GAD-65 expression or function has been investigated in the context of neurological and neuropsychiatric phenotypes linked to disrupted GABAergic neurotransmission, including seizure susceptibility and inhibitory interneuron dysfunction.

GAD-65 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous GAD2 expression without altering the underlying DNA sequence.

GAD-65 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the GAD2 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 GAD2 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous GAD-65 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native GAD2 locus and enabling the study of GAD-65-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of GAD-65 pathway restoration in tumor cells with silenced or reduced GAD2 expression.

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