HSPA8/HSC70 CRISPR Activation Plasmid (h): sc-418508-ACT

HSPA8 encodes heat shock cognate 71 kDa protein (HSC70), a constitutively expressed HSP70-family chaperone that supports proteostasis by mediating ATP-dependent folding, refolding, and trafficking of client proteins. HSC70 participates in clathrin-mediated endocytosis, chaperone-mediated autophagy via LAMP2A recognition, and protein quality control pathways linked to the ubiquitin–proteasome system and stress granule dynamics. Through regulation of nascent polypeptide handling, organelle protein import, and chaperone–cochaperone networks, HSPA8 influences cell survival under proteotoxic and metabolic stress. Dysregulation of HSC70-centered proteostasis is implicated in neurodegeneration, cancer cell stress adaptation, and viral infection biology, making HSPA8 a useful node for mechanistic studies of stress and trafficking pathways.

HSPA8/HSC70 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous HSPA8 expression without altering the underlying DNA sequence.

HSPA8/HSC70 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the HSPA8 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 HSPA8 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous HSPA8/HSC70 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native HSPA8 locus and enabling the study of HSPA8/HSC70-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of HSPA8/HSC70 pathway restoration in tumor cells with silenced or reduced HSPA8 expression.

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