Ataxin-3 CRISPR Activation Plasmid (h): sc-417498-ACT

ATXN3 encodes ataxin-3, a deubiquitinating enzyme that edits ubiquitin chains and helps regulate protein quality control through the ubiquitin–proteasome system and autophagy-linked pathways. Ataxin-3 participates in proteostasis and cellular stress responses by modulating the stability and turnover of misfolded or aggregation-prone substrates, with functional links to transcriptional regulation and DNA damage-associated signaling. Expanded polyglutamine tracts in ATXN3 are associated with spinocerebellar ataxia type 3 (Machado–Joseph disease), a neurodegenerative disorder characterized by protein aggregation and neuronal vulnerability. These properties make ATXN3 a useful node for studying ubiquitin signaling, aggregate biology, and pathways governing neuronal homeostasis.

Ataxin-3 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous ATXN3 expression without altering the underlying DNA sequence.

Ataxin-3 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the ATXN3 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 ATXN3 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Ataxin-3 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native ATXN3 locus and enabling the study of Ataxin-3-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Ataxin-3 pathway restoration in tumor cells with silenced or reduced ATXN3 expression.

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