GRAMD1B CRISPR Activation Plasmid (h): sc-408929-ACT

Human GRAMD1B encodes a GRAM domain–containing membrane-associated protein implicated in intracellular sterol transport and the organization of endoplasmic reticulum–plasma membrane contact sites. Through its GRAM domain–mediated lipid sensing and recruitment to specific membrane compartments, GRAMD1B is linked to cholesterol homeostasis, membrane composition, and lipid-dependent signaling processes that influence vesicular trafficking and cellular stress responses. Altered GRAMD1B expression has been reported in genomic and transcriptomic studies across multiple disease contexts, including cancer and immune-related conditions, supporting its use in mechanistic studies of lipid metabolism–coupled pathways. Functional interrogation of GRAMD1B helps clarify how sterol distribution and membrane contact dynamics modulate cell-state regulation and signaling network outputs.

GRAMD1B CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous GRAMD1B expression without altering the underlying DNA sequence.

GRAMD1B CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the GRAMD1B 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 GRAMD1B transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous GRAMD1B expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native GRAMD1B locus and enabling the study of GRAMD1B-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of GRAMD1B pathway restoration in tumor cells with silenced or reduced GRAMD1B expression.

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