CAR CRISPR Activation Plasmid (h): sc-418548-ACT

CXADR encodes the coxsackievirus and adenovirus receptor (CAR), an immunoglobulin superfamily adhesion molecule enriched at tight junctions where it contributes to epithelial barrier organization and intercellular contacts. CAR participates in junctional assembly and signaling processes linked to cytoskeletal remodeling and cell polarity, influencing cell–cell communication and tissue architecture. Altered CXADR expression and localization have been associated with changes in epithelial integrity, developmental programs, and tumor-associated phenotypes such as dysregulated adhesion and invasiveness. As a viral attachment factor, CAR is also relevant to studies of host–pathogen interactions and viral entry mechanisms in human cells.

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

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

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