VPS28 CRISPR Activation Plasmid (h): sc-404226-ACT

Human VPS28 encodes a core subunit of the ESCRT-I complex that coordinates endosomal sorting of ubiquitinated membrane proteins and promotes multivesicular body biogenesis en route to lysosomal degradation. Through ESCRT pathway activity, VPS28 helps regulate receptor downmodulation, membrane remodeling, and trafficking events that influence signal attenuation and proteostasis. ESCRT-I function also interfaces with cytokinetic abscission and viral budding processes that exploit host membrane scission machinery. Dysregulation of endolysosomal trafficking and ESCRT components is associated with altered growth factor signaling and cellular stress phenotypes relevant to cancer and neurodegeneration research.

VPS28 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous VPS28 expression without altering the underlying DNA sequence.

VPS28 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the VPS28 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 VPS28 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous VPS28 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native VPS28 locus and enabling the study of VPS28-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of VPS28 pathway restoration in tumor cells with silenced or reduced VPS28 expression.

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