EAAT3 CRISPR Activation Plasmid (h): sc-401539-ACT

SLC1A1 encodes the excitatory amino acid transporter 3 (EAAT3), a high-affinity sodium-dependent glutamate and aspartate transporter that helps regulate extracellular excitatory amino acid levels and supports intracellular glutamate availability for metabolism and redox balance. EAAT3 activity contributes to neurotransmitter clearance, synaptic signaling homeostasis, and neuronal vulnerability to oxidative stress through glutamate-dependent pathways that intersect with cysteine uptake and glutathione synthesis. In human tissues, SLC1A1 is linked to neuronal and epithelial transport processes and has been studied in the context of altered excitatory neurotransmission and cellular stress responses. Genetic and expression changes in SLC1A1 have been associated with neuropsychiatric and neurodevelopmental phenotypes, motivating mechanistic studies of transporter regulation and downstream signaling.

EAAT3 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous SLC1A1 expression without altering the underlying DNA sequence.

EAAT3 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the SLC1A1 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 SLC1A1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous EAAT3 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native SLC1A1 locus and enabling the study of EAAT3-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of EAAT3 pathway restoration in tumor cells with silenced or reduced SLC1A1 expression.

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