USP25 CRISPR Activation Plasmid (m): sc-424423-ACT

Mouse Usp25 encodes ubiquitin-specific peptidase 25 (USP25), a deubiquitinating enzyme that edits ubiquitin chains to modulate protein stability, trafficking, and signal termination. USP25 activity influences proteostasis and inflammatory signaling, including regulation of innate immune outputs linked to NF-κB and interferon-associated pathways through deubiquitination of key signaling components. In neural and immune contexts, Usp25 dosage has been connected to altered synaptic and inflammatory homeostasis, supporting investigation of its roles in neurodevelopmental phenotypes and neuroinflammation. Dysregulated ubiquitin signaling involving USP25 has also been studied in settings of proteotoxic stress and tumor-associated signaling rewiring, making it relevant to pathway-level mechanistic studies.

USP25 CRISPR Activation Plasmid (m) provides a targeted, non-destructive approach to upregulating endogenous Usp25 expression without altering the underlying DNA sequence.

USP25 CRISPR Activation Plasmid (m) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the Usp25 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 Usp25 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous USP25 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native Usp25 locus and enabling the study of USP25-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of USP25 pathway restoration in tumor cells with silenced or reduced Usp25 expression.

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