V-ATPase B1 CRISPR Activation Plasmid (h): sc-400926-ACT

ATP6V1B1 encodes the B1 subunit of the V1 catalytic domain of vacuolar H+-ATPase (V-ATPase), a multi-subunit proton pump that hydrolyzes ATP to acidify endosomes, lysosomes, and secretory vesicles. V-ATPase-driven luminal acidification supports receptor-mediated endocytosis, lysosomal enzyme maturation, autophagic flux, and vesicular trafficking, while also contributing to membrane proton transport processes that shape cellular pH homeostasis. In human physiology, ATP6V1B1 is enriched in specialized epithelia where regulated proton secretion and organelle acidification are critical. Dysregulation of V-ATPase subunits, including ATP6V1B1, is implicated in disorders of epithelial ion handling and lysosomal dysfunction-associated phenotypes, making it relevant for mechanistic studies of acidification-dependent signaling and proteostasis pathways.

V-ATPase B1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous ATP6V1B1 expression without altering the underlying DNA sequence.

V-ATPase B1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the ATP6V1B1 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 ATP6V1B1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous V-ATPase B1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native ATP6V1B1 locus and enabling the study of V-ATPase B1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of V-ATPase B1 pathway restoration in tumor cells with silenced or reduced ATP6V1B1 expression.

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