VPS16 CRISPR Activation Plasmid (h): sc-403914-ACT

Human VPS16 encodes a core subunit of the CORVET/HOPS tethering complexes that coordinate endosome and lysosome membrane fusion events. Through interactions with Rab GTPases and SNARE machinery, VPS16 supports endolysosomal trafficking, cargo sorting, and autophagosome–lysosome fusion, processes central to cellular proteostasis and receptor turnover. Perturbation of these pathways is linked to altered signaling homeostasis and neurodevelopmental and neurodegenerative disease mechanisms associated with lysosomal dysfunction. VPS16 is therefore widely used as a molecular handle to study vesicle tethering, lysosome biogenesis, and stress-responsive autophagy in human cell models.

VPS16 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous VPS16 expression without altering the underlying DNA sequence.

VPS16 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the VPS16 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 VPS16 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous VPS16 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native VPS16 locus and enabling the study of VPS16-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of VPS16 pathway restoration in tumor cells with silenced or reduced VPS16 expression.

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