USP4 CRISPR Activation Plasmid (h): sc-403543-ACT

Ubiquitin specific peptidase 4 (USP4) is a deubiquitinating enzyme that regulates protein stability and signaling output by removing ubiquitin chains from target substrates. In human cells, USP4 modulates key pathways including TGF-β/SMAD signaling, NF-κB–linked inflammatory responses, and receptor trafficking, thereby influencing transcriptional programs, proteostasis, and stress-adaptive processes. Through its effects on ubiquitin-dependent turnover, USP4 contributes to the control of cell proliferation, apoptosis, and immune signaling dynamics. Dysregulated USP4 activity or expression has been associated with altered signaling homeostasis in cancer biology and inflammatory disease mechanisms, making it a relevant node for pathway interrogation.

USP4 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous USP4 expression without altering the underlying DNA sequence.

USP4 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the USP4 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 USP4 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous USP4 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native USP4 locus and enabling the study of USP4-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of USP4 pathway restoration in tumor cells with silenced or reduced USP4 expression.

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