CRB3 CRISPR Activation Plasmid (h): sc-403145-ACT

CRB3 (crumbs homolog 3) encodes an apical polarity protein that localizes to tight junction–associated complexes and helps organize epithelial cell architecture. By supporting apical–basal polarity, junctional integrity, and vesicle trafficking, CRB3 contributes to processes such as lumen formation, ciliogenesis, and regulated proliferation through polarity-associated signaling networks. Altered CRB3 expression or localization has been linked to loss of epithelial organization and disrupted tissue homeostasis, features frequently examined in studies of tumor progression and metastasis. As a conserved polarity determinant, CRB3 is widely used as a molecular handle to interrogate epithelial differentiation and barrier function in human cell models.

CRB3 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous CRB3 expression without altering the underlying DNA sequence.

CRB3 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the CRB3 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 CRB3 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous CRB3 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native CRB3 locus and enabling the study of CRB3-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of CRB3 pathway restoration in tumor cells with silenced or reduced CRB3 expression.

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