GARNL1 CRISPR Activation Plasmid (h): sc-403936-ACT

Human RALGAPA1 encodes the GARNL1 subunit of the Ral GTPase-activating protein complex, a key negative regulator of RalA and RalB signaling downstream of Ras-family inputs. By accelerating GTP hydrolysis on Ral proteins, GARNL1 helps tune processes including vesicle trafficking, exocytosis, cytoskeletal remodeling, and cell-cycle–linked signaling nodes that influence cell growth and migration. Dysregulated Ral pathway activity is frequently implicated in oncogenic signaling networks and altered membrane dynamics, making RALGAPA1/GARNL1 a useful entry point for dissecting Ras–Ral axis modulation. Perturbation of this regulatory module is also relevant to studying pathway cross-talk with MAPK and PI3K signaling and context-dependent changes in cellular adhesion and polarity.

GARNL1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous RALGAPA1 expression without altering the underlying DNA sequence.

GARNL1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the RALGAPA1 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 RALGAPA1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous GARNL1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native RALGAPA1 locus and enabling the study of GARNL1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of GARNL1 pathway restoration in tumor cells with silenced or reduced RALGAPA1 expression.

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