EphA3 CRISPR Activation Plasmid (h): sc-401565-ACT

EPHA3 encodes the EphA3 receptor tyrosine kinase, a member of the ephrin–Eph family that mediates contact-dependent signaling to regulate cell positioning, adhesion, and tissue boundary formation. Upon binding membrane-tethered ephrin-A ligands, EphA3 initiates bidirectional signaling that remodels the actin cytoskeleton and modulates pathways controlling cell migration and guidance, including Rho family GTPase-dependent programs and downstream kinase signaling. EphA3 activity is implicated in developmental processes such as axon guidance and vascular patterning, and altered EPHA3 expression or signaling has been associated with tumor cell invasion, microenvironmental interactions, and oncogenic pathway rewiring in multiple cancer contexts. These features make EPHA3 a useful target for dissecting receptor-driven communication networks and context-specific phenotypic plasticity in human cells.

EphA3 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous EPHA3 expression without altering the underlying DNA sequence.

EphA3 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the EPHA3 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 EPHA3 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous EphA3 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native EPHA3 locus and enabling the study of EphA3-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of EphA3 pathway restoration in tumor cells with silenced or reduced EPHA3 expression.

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