V-ATPase B2 CRISPR Activation Plasmid (h): sc-401896-ACT

ATP6V1B2 encodes the B2 subunit of the V1 peripheral domain of vacuolar H+-ATPase (V-ATPase), a multisubunit proton pump that hydrolyzes ATP to acidify endosomes, lysosomes, and other intracellular compartments. This organellar acidification is essential for receptor-mediated endocytosis, autophagic flux, lysosomal enzyme activation, and vesicular trafficking, and it indirectly shapes nutrient sensing and mTOR-linked metabolic adaptation. By regulating luminal pH, V-ATPase activity influences protein processing and turnover, antigen processing, and membrane protein recycling. Dysregulated vesicle and lysosome acidification pathways involving V-ATPase components have been associated with neurodevelopmental and neurological phenotypes and broader disorders of intracellular trafficking, making ATP6V1B2 a useful node for studying proteostasis and endolysosomal dysfunction.

V-ATPase B2 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous ATP6V1B2 expression without altering the underlying DNA sequence.

V-ATPase B2 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the ATP6V1B2 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 ATP6V1B2 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous V-ATPase B2 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native ATP6V1B2 locus and enabling the study of V-ATPase B2-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of V-ATPase B2 pathway restoration in tumor cells with silenced or reduced ATP6V1B2 expression.

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