OTUD4 CRISPR Activation Plasmid (h): sc-412322-ACT

OTUD4 encodes a human ovarian tumor domain–containing deubiquitinase that regulates ubiquitin-dependent protein turnover and signaling by removing ubiquitin chains from substrate proteins. Through modulation of ubiquitination dynamics, OTUD4 influences proteostasis, stress-response pathways, and context-dependent control of innate immune and inflammatory signaling. Altered OTUD4 activity has been linked in the literature to dysregulated ubiquitin signaling networks that intersect with genome stability and cell fate decisions, making it relevant for mechanistic studies in cancer biology and neurobiology. Its enzymatic role in deubiquitination also positions OTUD4 as a useful node for dissecting pathway crosstalk between ubiquitin remodeling and transcriptional programs.

OTUD4 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous OTUD4 expression without altering the underlying DNA sequence.

OTUD4 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the OTUD4 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 OTUD4 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous OTUD4 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native OTUD4 locus and enabling the study of OTUD4-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of OTUD4 pathway restoration in tumor cells with silenced or reduced OTUD4 expression.

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