NORRIN CRISPR Activation Plasmid (h): sc-405323-ACT

Human NDP encodes the secreted protein NORRIN, an atypical Wnt ligand that binds Frizzled-4 and LRP5/6 to activate canonical Wnt/β-catenin signaling. This pathway supports vascular development and maintenance in the retina and inner ear, influencing endothelial–pericyte interactions, barrier integrity, and neurovascular homeostasis. Dysregulated NDP/NORRIN signaling is linked to inherited vitreoretinopathies and retinal vascular defects, and is frequently studied in the context of angiogenic remodeling and neurodevelopmental signaling networks. Experimental modulation of NDP expression provides a tractable approach to dissect Wnt pathway crosstalk, transcriptional programs downstream of β-catenin, and cell-type–specific responses in ocular and vascular models.

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

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

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