V-ATPase D1 CRISPR Activation Plasmid (h): sc-403669-ACT

ATP6V0D1 encodes the D1 subunit of the V-type proton ATPase (V-ATPase) V0 membrane sector, a core component of the rotary proton pump that acidifies endosomes, lysosomes, and other intracellular organelles. Through organelle acidification, V-ATPase activity supports receptor-mediated endocytosis, lysosomal proteolysis, autophagy, and vesicular trafficking, and it contributes to pH homeostasis across multiple cell types. Proper V-ATPase assembly and proton translocation are also important for endolysosomal signaling platforms, including pathways that depend on lysosome function such as mTORC1 nutrient sensing. Dysregulation of endolysosomal acidification and trafficking is linked to neurodegeneration, altered immune responses, and cancer-associated invasion phenotypes, making ATP6V0D1 a relevant target for mechanistic studies of organelle function.

V-ATPase D1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous ATP6V0D1 expression without altering the underlying DNA sequence.

V-ATPase D1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the ATP6V0D1 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 ATP6V0D1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous V-ATPase D1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native ATP6V0D1 locus and enabling the study of V-ATPase D1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of V-ATPase D1 pathway restoration in tumor cells with silenced or reduced ATP6V0D1 expression.

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