UBPY CRISPR Activation Plasmid (h): sc-403737-ACT

Human USP8 encodes the deubiquitinating enzyme UBPY, a ubiquitin-specific protease that edits ubiquitin chains on endocytic trafficking components and receptor substrates to control protein stability and sorting. UBPY acts in the endosomal system in coordination with ESCRT-associated machinery, regulating cargo deubiquitination, receptor recycling versus lysosomal degradation, and downstream signaling amplitude. Through these activities, USP8 influences pathways linked to growth factor receptor signaling, vesicle transport, and proteostasis, which are frequently implicated in dysregulated cell signaling and altered endolysosomal function. Genetic and functional studies have connected USP8 perturbation with disease-relevant phenotypes involving aberrant receptor turnover and signaling network rewiring, supporting its utility as a mechanistic node in cellular homeostasis research.

UBPY CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous USP8 expression without altering the underlying DNA sequence.

UBPY CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the USP8 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 USP8 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous UBPY expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native USP8 locus and enabling the study of UBPY-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of UBPY pathway restoration in tumor cells with silenced or reduced USP8 expression.

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