VPS4B CRISPR Activation Plasmid (h): sc-403260-ACT

VPS4B encodes a AAA+ ATPase that powers ESCRT-III disassembly and recycling, a key step in endosomal sorting, multivesicular body biogenesis, and lysosomal degradation of membrane proteins. By coordinating membrane remodeling events, VPS4B contributes to receptor downregulation, cytokinesis, plasma membrane repair, and the budding of certain enveloped viruses. Perturbation of ESCRT–VPS4 dynamics can alter cargo trafficking and signaling outputs, linking VPS4B-associated pathways to cellular homeostasis and stress responses relevant to diverse disease biology. As a core regulator of vesicle-mediated transport, VPS4B is frequently studied in the context of proteostasis, membrane integrity, and trafficking-dependent signaling networks.

VPS4B CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous VPS4B expression without altering the underlying DNA sequence.

VPS4B CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the VPS4B 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 VPS4B transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous VPS4B expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native VPS4B locus and enabling the study of VPS4B-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of VPS4B pathway restoration in tumor cells with silenced or reduced VPS4B expression.

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