GRP94 CRISPR Activation Plasmid (m): sc-423491-ACT

Mouse Hsp90b1 encodes GRP94 (gp96), an endoplasmic reticulum–resident HSP90 family chaperone that supports protein folding, quality control, and ER homeostasis. GRP94 is essential for the maturation and trafficking of select secretory and membrane proteins, including integrins and Toll-like receptors, thereby influencing cell adhesion, innate immune signaling, and receptor-mediated pathways. Its activity is closely coupled to ER stress signaling and the unfolded protein response, linking Hsp90b1 regulation to proteostasis, inflammation, and cellular adaptation to stress. Dysregulated GRP94 function has been associated with altered immune phenotypes, disrupted secretory pathway biology, and disease-relevant stress responses in models of cancer and metabolic dysfunction.

GRP94 CRISPR Activation Plasmid (m) provides a targeted, non-destructive approach to upregulating endogenous Hsp90b1 expression without altering the underlying DNA sequence.

GRP94 CRISPR Activation Plasmid (m) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the Hsp90b1 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 Hsp90b1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous GRP94 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native Hsp90b1 locus and enabling the study of GRP94-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of GRP94 pathway restoration in tumor cells with silenced or reduced Hsp90b1 expression.

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