VPS25 CRISPR Activation Plasmid (h): sc-404109-ACT

Human VPS25 encodes a core subunit of the ESCRT-II complex that coordinates endosomal sorting and multivesicular body biogenesis, enabling ubiquitinated membrane proteins to be trafficked toward lysosomal degradation. Through ESCRT-dependent membrane remodeling, VPS25 contributes to receptor downregulation, membrane protein homeostasis, and broader control of signaling intensity and duration. ESCRT machinery also interfaces with cytokinesis and autophagy-related membrane dynamics, linking VPS25 function to cell division and stress-adaptive proteostasis. Dysregulation of endosomal-lysosomal trafficking and ESCRT pathway components is associated with altered growth factor signaling, impaired protein turnover, and neurodegenerative and cancer-relevant cellular phenotypes.

VPS25 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous VPS25 expression without altering the underlying DNA sequence.

VPS25 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the VPS25 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 VPS25 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous VPS25 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native VPS25 locus and enabling the study of VPS25-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of VPS25 pathway restoration in tumor cells with silenced or reduced VPS25 expression.

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