eEF2K CRISPR Activation Plasmid (h): sc-402166-ACT

EEF2K encodes eukaryotic elongation factor 2 kinase (eEF2K), a Ca2+/calmodulin-dependent kinase that phosphorylates eEF2 to slow ribosomal translocation and tune protein synthesis rates during cellular stress. This signaling node integrates nutrient and energy sensing via AMPK and mTORC1-linked pathways, coupling translational control to autophagy, metabolic adaptation, and cell-cycle regulation. In human cells, eEF2K activity influences survival under hypoxia and nutrient limitation and has been studied in contexts of tumor biology and neurobiology where proteostasis and synaptic plasticity are perturbed. Dysregulated EEF2K signaling is associated with altered growth control and stress tolerance, making it relevant for dissecting translation-dependent phenotypes.

eEF2K CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous EEF2K expression without altering the underlying DNA sequence.

eEF2K CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the EEF2K 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 EEF2K transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous eEF2K expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native EEF2K locus and enabling the study of eEF2K-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of eEF2K pathway restoration in tumor cells with silenced or reduced EEF2K expression.

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