V-ATPase D CRISPR Activation Plasmid (h): sc-403291-ACT

ATP6V1D encodes the D subunit of the vacuolar H+-ATPase (V-ATPase) V1 domain, a rotary ATPase that couples ATP hydrolysis to proton pumping across intracellular membranes. By supporting organelle acidification, V-ATPase D contributes to endosome–lysosome maturation, autophagic flux, receptor recycling, and pH-dependent trafficking through the secretory and endocytic pathways. V-ATPase-dependent pH control also influences nutrient sensing, including mTORC1 signaling at the lysosomal surface, and shapes the activity of proteases within acidic compartments. Dysregulated V-ATPase function and vesicular acidification have been linked to altered proteostasis, neurodegenerative processes, and cancer-associated metabolic and invasive phenotypes, making ATP6V1D a useful node for mechanistic studies of cellular homeostasis.

V-ATPase D CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous ATP6V1D expression without altering the underlying DNA sequence.

V-ATPase D CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the ATP6V1D 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 ATP6V1D transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous V-ATPase D expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native ATP6V1D locus and enabling the study of V-ATPase D-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of V-ATPase D pathway restoration in tumor cells with silenced or reduced ATP6V1D expression.

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