ATP7A CRISPR Activation Plasmid (h): sc-402387-ACT

ATP7A encodes a copper-transporting P-type ATPase that regulates intracellular copper distribution by exporting copper from the cytosol and delivering it to the secretory pathway for metallation of cuproenzymes. By controlling copper efflux and trafficking, ATP7A supports redox homeostasis, mitochondrial and oxidative stress responses, and enzyme maturation within the Golgi network. Dysregulated ATP7A activity perturbs copper balance and impacts processes such as connective tissue formation and neurodevelopmental signaling through impaired cuproenzyme function. Consequently, ATP7A is widely studied in models of copper metabolism disorders and cellular stress pathways linked to copper-dependent enzymatic activity.

ATP7A CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous ATP7A expression without altering the underlying DNA sequence.

ATP7A CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the ATP7A 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 ATP7A transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous ATP7A expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native ATP7A locus and enabling the study of ATP7A-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of ATP7A pathway restoration in tumor cells with silenced or reduced ATP7A expression.

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