nephrocystin-2 CRISPR Activation Plasmid (h): sc-403310-ACT

INVS encodes nephrocystin-2 (inversin), a cilia-associated protein that localizes to primary cilia and basal bodies and contributes to centrosome organization and ciliary signaling. Nephrocystin-2 participates in planar cell polarity and modulates Wnt pathway outputs, supporting the balance between canonical β-catenin signaling and non-canonical signaling during tissue morphogenesis. Disruption of INVS perturbs cilia-dependent mechanosensation and epithelial polarity, processes central to renal tubular development and homeostasis. Genetic variation in INVS is linked to ciliopathy phenotypes, including nephronophthisis spectrum disease and laterality defects, making it a useful target for studying cilia-driven signaling networks in human cells.

nephrocystin-2 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous INVS expression without altering the underlying DNA sequence.

nephrocystin-2 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the INVS 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 INVS transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous nephrocystin-2 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native INVS locus and enabling the study of nephrocystin-2-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of nephrocystin-2 pathway restoration in tumor cells with silenced or reduced INVS expression.

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