ATP6H CRISPR Activation Plasmid (h): sc-411234-ACT

ATP6V0E1 encodes ATP6H, a core component of the V0 domain of vacuolar H⁺-ATPase (V-ATPase) that drives proton translocation required for acidification of endosomes, lysosomes, and secretory vesicles. By controlling organelle pH, V-ATPase activity regulates receptor-mediated endocytosis, protein sorting and degradation, autophagic flux, and membrane trafficking linked to mTOR and lysosome-dependent signaling. Perturbation of V-ATPase subunits disrupts vesicular homeostasis and can alter antigen processing, neurotransmitter loading, and cellular metabolism. Dysregulated vesicle acidification and lysosomal function are implicated in cancer cell adaptation, neurodegenerative phenotypes, and immune dysfunction, supporting ATP6V0E1 as a useful node for studying endolysosomal pathways.

ATP6V0E1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous ATP6V0E1 expression without altering the underlying DNA sequence.

ATP6V0E1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the ATP6V0E1 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 ATP6V0E1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous ATP6V0E1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native ATP6V0E1 locus and enabling the study of ATP6V0E1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of ATP6V0E1 pathway restoration in tumor cells with silenced or reduced ATP6V0E1 expression.

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