VPS4A CRISPR Activation Plasmid (h): sc-402005-ACT

Human VPS4A encodes an AAA+ ATPase that powers ESCRT-III disassembly and recycling, enabling membrane remodeling events required for multivesicular body biogenesis, endosomal cargo sorting, cytokinetic abscission, and plasma membrane repair. VPS4A activity coordinates with ESCRT components to regulate receptor downregulation, exosome and microvesicle biogenesis, and nuclear envelope sealing after mitosis. Through its central role in endolysosomal trafficking and membrane fission, VPS4A is frequently studied in pathways impacting proteostasis, innate immune signaling, and viral budding that hijacks ESCRT machinery. Dysregulation of ESCRT/VPS4 functions has been linked to neurodegenerative phenotypes, altered growth factor signaling, and cancer-associated changes in vesicle trafficking and cell division.

VPS4A CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous VPS4A expression without altering the underlying DNA sequence.

VPS4A CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the VPS4A 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 VPS4A transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous VPS4A expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native VPS4A locus and enabling the study of VPS4A-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of VPS4A pathway restoration in tumor cells with silenced or reduced VPS4A expression.

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