DDEF2 CRISPR Activation Plasmid (h): sc-403676-ACT

Human ASAP2 (also known as DDEF2) encodes an ARF GTPase-activating protein that localizes to focal adhesions and interfaces with integrin-linked signaling to coordinate actin remodeling, cell adhesion, and directional migration. Through regulation of ARF-family small GTPases, DDEF2 influences membrane trafficking and cytoskeletal dynamics that shape cell spreading and invasive behavior. These processes connect ASAP2/DDEF2 activity to pathways commonly interrogated in oncogenic transformation, metastasis-associated motility, and other disorders involving aberrant adhesion and tissue remodeling. ASAP2 is therefore frequently studied in contexts such as focal adhesion turnover, endocytosis, and signaling crosstalk between ARF and Rho-family networks.

DDEF2 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous ASAP2 expression without altering the underlying DNA sequence.

DDEF2 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the ASAP2 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 ASAP2 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous DDEF2 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native ASAP2 locus and enabling the study of DDEF2-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of DDEF2 pathway restoration in tumor cells with silenced or reduced ASAP2 expression.

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