GRASP65 CRISPR Activation Plasmid (h): sc-402436-ACT

Human GORASP1 encodes GRASP65, a peripheral membrane protein that localizes to the cis-Golgi and functions as a key organizer of Golgi ribbon architecture. GRASP65 contributes to Golgi stacking, vesicle tethering, and reassembly of Golgi membranes during mitotic entry and exit, integrating with ER-to-Golgi trafficking and broader secretory pathway control. Through its roles in organelle organization and cargo processing, GRASP65 influences protein sorting, glycosylation, and polarized transport that are central to cell-cycle progression and stress responses. Dysregulation of Golgi structure and trafficking pathways involving GRASP65 is relevant to disease-associated phenotypes in cancer biology and neurodegeneration where altered secretion and organelle remodeling are commonly observed.

GRASP65 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous GORASP1 expression without altering the underlying DNA sequence.

GRASP65 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the GORASP1 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 GORASP1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous GRASP65 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native GORASP1 locus and enabling the study of GRASP65-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of GRASP65 pathway restoration in tumor cells with silenced or reduced GORASP1 expression.

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