V-ATPase α1 CRISPR Activation Plasmid (h): sc-403882-ACT

ATP6V1A encodes the V-ATPase α1 subunit, a core component of the cytosolic V1 sector that couples ATP hydrolysis to proton translocation by the vacuolar H⁺-ATPase complex. This enzyme acidifies endosomes, lysosomes, and secretory vesicles, supporting receptor-mediated endocytosis, autophagic flux, membrane trafficking, and protein processing, while also contributing to pH homeostasis at the plasma membrane in specialized contexts. By controlling organelle acidification, V-ATPase activity influences nutrient sensing and signaling programs, including pathways linked to mTORC1 regulation and vesicle maturation. Dysregulated vacuolar acidification and altered ATP6V1A function have been associated with phenotypes relevant to neurobiology, cancer cell biology, and host–pathogen interactions, making it a useful target for mechanistic studies of proteostasis and intracellular transport.

V-ATPase α1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous ATP6V1A expression without altering the underlying DNA sequence.

V-ATPase α1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the ATP6V1A 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 ATP6V1A transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous V-ATPase α1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native ATP6V1A locus and enabling the study of V-ATPase α1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of V-ATPase α1 pathway restoration in tumor cells with silenced or reduced ATP6V1A expression.

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