Gads CRISPR Activation Plasmid (h): sc-402831-ACT

GRAP2 encodes the adaptor protein Gads, a hematopoietic signaling scaffold that links phosphorylated LAT to SLP-76 to assemble the T cell receptor (TCR) signalosome. Through its SH2 and SH3 domains, Gads coordinates recruitment of effector complexes that promote PLCγ1 activation, calcium flux, MAPK signaling, and downstream transcriptional programs controlling T cell activation, differentiation, and cytokine production. GRAP2 function is also relevant to immune receptor signaling in other leukocyte lineages and influences cytoskeletal remodeling and immunological synapse formation. Dysregulated GRAP2/Gads-dependent signaling has been associated with altered immune responses and is studied in contexts such as immune deficiency, autoimmunity-related mechanisms, and hematologic malignancy signaling networks.

Gads CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous GRAP2 expression without altering the underlying DNA sequence.

Gads CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the GRAP2 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 GRAP2 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Gads expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native GRAP2 locus and enabling the study of Gads-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Gads pathway restoration in tumor cells with silenced or reduced GRAP2 expression.

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