AF-6 CRISPR Activation Plasmid (h2): sc-404503-ACT-2

Human AFDN encodes the junctional scaffold protein AF-6 (afadin), a multidomain adaptor that links nectin family adhesion receptors to the actin cytoskeleton to organize adherens junctions and maintain epithelial and endothelial barrier integrity. AF-6 coordinates cytoskeletal remodeling and cell polarity by integrating signals from small GTPases such as Rap1 and Ras, contributing to contact formation, collective migration, and mechanotransduction in tissue morphogenesis. Dysregulated AFDN/AF-6 function and localization have been associated with altered cell–cell adhesion and invasive behavior in cancer, and variants affecting junctional stability have been implicated in neurodevelopmental and vascular phenotypes. AFDN is therefore widely used in gene editing and functional genomics studies to interrogate junction assembly, polarity networks, and signaling crosstalk at cell–cell contacts in human cellular models.

AF-6 CRISPR Activation Plasmid (h2) provides a targeted, non-destructive approach to upregulating endogenous AFDN expression without altering the underlying DNA sequence.

AF-6 CRISPR Activation Plasmid (h2) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the AFDN 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 AFDN transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous AF-6 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native AFDN locus and enabling the study of AF-6-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of AF-6 pathway restoration in tumor cells with silenced or reduced AFDN expression.

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