nicastrin CRISPR Activation Plasmid (h): sc-402790-ACT

Human NCSTN encodes nicastrin, a type I transmembrane glycoprotein that serves as an essential subunit of the γ-secretase complex, supporting substrate recognition and maturation of presenilin-containing protease assemblies. Through γ-secretase activity, nicastrin contributes to regulated intramembrane proteolysis of diverse substrates including NOTCH receptors and amyloid precursor protein, linking NCSTN to Notch signaling, cell fate decisions, and membrane protein turnover. Perturbation of γ-secretase components can alter differentiation programs, inflammatory signaling, and proteostasis, making NCSTN a widely studied node in pathways relevant to neurobiology, epithelial homeostasis, and skin biology. NCSTN dysregulation and variants have been associated with disorders involving aberrant Notch pathway activity and altered epidermal or immune responses, providing mechanistic context for functional genomics studies.

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

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

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