V-ATPase G1 CRISPR Activation Plasmid (h): sc-406125-ACT

ATP6V1G1 encodes the G1 subunit of the V1 peripheral domain of vacuolar H+-ATPase (V-ATPase), a multi-subunit proton pump that couples ATP hydrolysis to organelle acidification. V-ATPase-driven pH control is central to endosome–lysosome maturation, receptor-mediated endocytosis, autophagic flux, and vesicular trafficking, and it influences signaling networks such as mTORC1 through lysosomal nutrient sensing. By modulating luminal acidity, V-ATPase activity affects protease activation, antigen processing, and membrane fusion dynamics across intracellular compartments. Dysregulated organellar pH homeostasis and V-ATPase subunit imbalance have been linked to altered metabolism, invasive phenotypes, and stress-adaptation programs observed in multiple disease-relevant cellular contexts.

V-ATPase G1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous ATP6V1G1 expression without altering the underlying DNA sequence.

V-ATPase G1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the ATP6V1G1 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 ATP6V1G1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous V-ATPase G1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native ATP6V1G1 locus and enabling the study of V-ATPase G1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of V-ATPase G1 pathway restoration in tumor cells with silenced or reduced ATP6V1G1 expression.

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