14-3-3 ε CRISPR Activation Plasmid (h): sc-400998-ACT

YWHAE encodes the human 14-3-3 epsilon (ε) adaptor protein, a phosphoserine/phosphothreonine-binding regulator that modulates the localization, stability, and activity of diverse client proteins. Through interactions with kinases and signaling intermediates, 14-3-3 ε helps coordinate PI3K/AKT, MAPK, and cell-cycle checkpoint signaling, influencing apoptosis, proliferation, and stress responses. It contributes to cytoskeletal remodeling and neuronal development pathways by scaffolding phosphoprotein complexes and tuning subcellular trafficking. Dysregulated YWHAE expression or altered 14-3-3 ε interactions have been associated with neurodevelopmental phenotypes and oncogenic signaling networks, supporting its use in mechanistic studies of signaling rewiring.

14-3-3 ε CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous YWHAE expression without altering the underlying DNA sequence.

14-3-3 ε CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the YWHAE 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 YWHAE transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous 14-3-3 ε expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native YWHAE locus and enabling the study of 14-3-3 ε-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of 14-3-3 ε pathway restoration in tumor cells with silenced or reduced YWHAE expression.

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