HYPE CRISPR Activation Plasmid (h): sc-406626-ACT

Human FICD encodes HYPE, an endoplasmic reticulum–localized Fic domain enzyme that catalyzes reversible AMPylation and de-AMPylation of protein substrates, prominently the HSPA5/BiP chaperone. Through modulation of BiP activity, HYPE shapes ER proteostasis, unfolded protein response (UPR) signaling, and adaptive stress programs that influence protein folding capacity and secretion. This regulatory axis links FICD to pathways controlling ER homeostasis, calcium handling, and cell survival under proteotoxic stress. Dysregulated ER stress and UPR signaling are implicated across neurodegeneration, metabolic dysfunction, and cancer-relevant phenotypes, making FICD/HYPE a useful node for mechanistic studies of proteostasis.

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

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

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