HAH1 CRISPR Activation Plasmid (h): sc-404870-ACT

ATOX1 encodes HAH1, a cytosolic copper chaperone that binds Cu(I) and delivers it to P-type ATPases ATP7A/ATP7B within the secretory pathway, supporting metallation of cuproenzymes and maintenance of cellular copper homeostasis. Through these trafficking steps, ATOX1 influences redox balance, mitochondrial function, and oxidative stress responses, and can indirectly modulate signaling networks linked to proliferation and migration. Dysregulated copper handling and altered ATOX1 activity have been associated with neurodegenerative and metabolic phenotypes, and are frequently explored in the context of copper-dependent tumor biology and inflammatory stress. Because copper availability shapes enzyme activity and transcriptional programs, ATOX1 is widely studied in pathways governing metal transport, protein maturation, and cellular adaptation to oxidative conditions.

HAH1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous ATOX1 expression without altering the underlying DNA sequence.

HAH1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the ATOX1 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 ATOX1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous HAH1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native ATOX1 locus and enabling the study of HAH1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of HAH1 pathway restoration in tumor cells with silenced or reduced ATOX1 expression.

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