Usherin CRISPR Activation Plasmid (h): sc-409699-ACT

USH2A encodes usherin, a large extracellular matrix–associated transmembrane protein enriched in sensory epithelia where it supports the architecture and mechanotransduction of photoreceptors and inner-ear hair cells. Usherin participates in the organization of Usher protein networks and contributes to cell–cell and cell–matrix interactions that stabilize stereocilia bundles and the periciliary membrane complex. Through these roles, USH2A is linked to processes governing ciliogenesis, adhesion, and trafficking in polarized cells. Genetic perturbation of USH2A is strongly associated with inherited retinal degeneration and syndromic hearing/vision phenotypes, making it a key locus for studying sensory cell maintenance and degeneration mechanisms.

Usherin CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous USH2A expression without altering the underlying DNA sequence.

Usherin CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the USH2A 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 USH2A transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Usherin expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native USH2A locus and enabling the study of Usherin-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Usherin pathway restoration in tumor cells with silenced or reduced USH2A expression.

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