NPC2 CRISPR Activation Plasmid (h): sc-402010-ACT

Human NPC2 encodes a soluble lysosomal cholesterol-binding protein that cooperates with NPC1 to mobilize unesterified cholesterol and other lipids from late endosomes/lysosomes to downstream trafficking routes. NPC2 activity supports cellular sterol homeostasis, membrane composition, and lipid-dependent signaling by regulating cholesterol egress and distribution to the endoplasmic reticulum and plasma membrane. Disruption of NPC2-dependent transport leads to lysosomal lipid accumulation and perturbs endolysosomal function, linking NPC2 to Niemann-Pick disease type C2 and broader pathways involved in neurodegeneration and metabolic dysfunction. As a core component of intracellular lipid trafficking, NPC2 is frequently studied in mechanisms governing autophagy-lysosome dynamics, vesicular transport, and cholesterol-regulated gene expression programs.

NPC2 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous NPC2 expression without altering the underlying DNA sequence.

NPC2 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the NPC2 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 NPC2 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous NPC2 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native NPC2 locus and enabling the study of NPC2-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of NPC2 pathway restoration in tumor cells with silenced or reduced NPC2 expression.

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