GAP1-InsP₄ BP CRISPR Activation Plasmid (h): sc-403002-ACT

RASA3 encodes the Ras GTPase-activating protein GAP1-InsP4 BP, a regulatory factor that accelerates GTP hydrolysis on Ras-family small GTPases to modulate signal amplitude and duration. Through its responsiveness to inositol polyphosphates, GAP1-InsP4 BP integrates phosphoinositide-linked second-messenger cues with Ras-dependent pathways controlling proliferation, differentiation, cytoskeletal dynamics, and vesicular trafficking. RASA3 activity is implicated in hematopoietic and vascular biology, where altered small GTPase regulation can perturb platelet function, endothelial signaling, and developmental programs. Dysregulation of Ras pathway control is broadly relevant to oncogenic signaling and other disorders characterized by aberrant MAPK/PI3K network activity, making RASA3 a useful node for mechanistic studies of signal transduction.

GAP1-InsP4 BP CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous RASA3 expression without altering the underlying DNA sequence.

GAP1-InsP4 BP CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the RASA3 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 RASA3 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous GAP1-InsP4 BP expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native RASA3 locus and enabling the study of GAP1-InsP4 BP-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of GAP1-InsP4 BP pathway restoration in tumor cells with silenced or reduced RASA3 expression.

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