ARHGEF5 CRISPR Activation Plasmid (h): sc-409563-ACT

ARHGEF5 encodes a Rho guanine nucleotide exchange factor that activates Rho-family GTPases, linking signals from cell-surface receptors to actin cytoskeleton remodeling. Through regulation of RhoA-driven pathways, ARHGEF5 influences cell adhesion, polarity, and motility, and contributes to focal adhesion dynamics and stress fiber formation. This signaling axis interfaces with pathways controlling proliferation and survival, making ARHGEF5 a useful node for studying cytoskeletal control and signal transduction. Altered Rho GTPase regulation has been associated with invasive phenotypes and aberrant tissue organization, supporting investigation of ARHGEF5 in disease-relevant models of dysregulated migration and growth.

ARHGEF5 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous ARHGEF5 expression without altering the underlying DNA sequence.

ARHGEF5 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the ARHGEF5 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 ARHGEF5 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous ARHGEF5 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native ARHGEF5 locus and enabling the study of ARHGEF5-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of ARHGEF5 pathway restoration in tumor cells with silenced or reduced ARHGEF5 expression.

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