NET-4 CRISPR Activation Plasmid (h): sc-404621-ACT

Human TSPAN5 encodes NET-4, a tetraspanin family membrane protein that organizes tetraspanin-enriched microdomains and modulates the composition and signaling output of partner receptors at the cell surface. By scaffolding adhesion and signaling complexes, NET-4 influences cell–cell communication, membrane trafficking, and cytoskeletal remodeling processes that shape neuronal network properties and other context-specific cellular behaviors. Altered tetraspanin network regulation has been linked to changes in synaptic function and neurodevelopmental phenotypes, making TSPAN5 a useful entry point for dissecting membrane microdomain–dependent signaling. NET-4 regulation is therefore relevant for studies of signaling pathway crosstalk, receptor dynamics, and genotype-to-phenotype relationships in human cell models.

NET-4 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous TSPAN5 expression without altering the underlying DNA sequence.

NET-4 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the TSPAN5 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 TSPAN5 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous NET-4 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native TSPAN5 locus and enabling the study of NET-4-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of NET-4 pathway restoration in tumor cells with silenced or reduced TSPAN5 expression.

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