Cingulin CRISPR Activation Plasmid (h): sc-404409-ACT

Human CGN encodes cingulin, a cytoplasmic adaptor concentrated at tight junctions where it links junctional membrane complexes to the cortical actin cytoskeleton. Cingulin helps organize epithelial and endothelial barrier function by coordinating junction assembly, cytoskeletal tension, and signaling crosstalk that influences cell polarity and paracellular permeability. Through interactions with tight junction scaffolds and Rho family GTPase regulatory circuits, CGN contributes to dynamic remodeling of cell–cell contacts during development, wound responses, and tissue homeostasis. Altered tight junction architecture and CGN-associated barrier dysfunction are recurrent features in inflammation, tumor progression, and epithelial–mesenchymal plasticity, motivating mechanistic studies in barrier-relevant disease models.

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

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

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