Aph-1 CRISPR Activation Plasmid (h): sc-403984-ACT

Human APH1A encodes Aph-1, an essential scaffold subunit of the γ-secretase complex that supports assembly and stability of the protease responsible for intramembrane cleavage of multiple type I membrane proteins. Through γ-secretase activity, Aph-1 contributes to regulated intramembrane proteolysis pathways, including processing of NOTCH receptors and amyloid precursor protein, thereby influencing cell fate decisions, differentiation programs, and neuronal signaling. Perturbation of γ-secretase composition or activity is linked to altered Notch pathway output and amyloidogenic processing, connecting APH1A-associated mechanisms to cancer biology, neurodegeneration, and developmental phenotypes. APH1A expression and complex stoichiometry are therefore relevant for studies of protease assembly, membrane trafficking, and signal transduction in human cells.

Aph-1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous APH1A expression without altering the underlying DNA sequence.

Aph-1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the APH1A 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 APH1A transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Aph-1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native APH1A locus and enabling the study of Aph-1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Aph-1 pathway restoration in tumor cells with silenced or reduced APH1A expression.

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