VAChT CRISPR Activation Plasmid (h): sc-401240-ACT

SLC18A3 encodes vesicular acetylcholine transporter (VAChT), a synaptic vesicle membrane protein that packages cytosolic acetylcholine into secretory vesicles in cholinergic neurons and other cholinergic cell types. By supplying vesicular acetylcholine for regulated exocytosis, VAChT is essential for synaptic transmission at neuromuscular junctions and throughout the autonomic and central nervous systems, influencing excitation–contraction coupling and neuronal circuit activity. VAChT function is tightly linked to acetylcholine metabolism and vesicle cycling pathways, including coordination with choline acetyltransferase-dependent synthesis and vesicular trafficking machinery. Altered cholinergic signaling and dysregulated acetylcholine release are implicated in multiple neurobiology contexts, making SLC18A3 a useful target for studying mechanisms underlying cholinergic dysfunction in disease-relevant models.

VAChT CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous SLC18A3 expression without altering the underlying DNA sequence.

VAChT CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the SLC18A3 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 SLC18A3 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous VAChT expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native SLC18A3 locus and enabling the study of VAChT-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of VAChT pathway restoration in tumor cells with silenced or reduced SLC18A3 expression.

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