VPS45 CRISPR Activation Plasmid (h): sc-411599-ACT

VPS45 encodes a conserved Sec1/Munc18 family regulator of vesicular trafficking that coordinates SNARE-dependent membrane fusion events required for endosome-to-Golgi transport and endosomal maturation. In human cells, VPS45 supports the stability and function of the HOPS/CORVET-related tethering machinery and helps maintain cargo sorting, receptor recycling, and lysosome-directed trafficking. Disruption of VPS45-dependent trafficking perturbs organelle homeostasis, impacts autophagy–lysosome pathways, and can alter signaling output by changing receptor availability at the plasma membrane. Genetic and functional studies link VPS45 dysfunction to immune and hematopoietic phenotypes, making it relevant for mechanistic research into intracellular transport defects and their downstream cellular consequences.

VPS45 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous VPS45 expression without altering the underlying DNA sequence.

VPS45 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the VPS45 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 VPS45 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous VPS45 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native VPS45 locus and enabling the study of VPS45-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of VPS45 pathway restoration in tumor cells with silenced or reduced VPS45 expression.

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