Visual Arrestin CRISPR Activation Plasmid (h): sc-402997-ACT

SAG encodes visual arrestin (arrestin-1), a photoreceptor-specific regulatory protein that terminates rhodopsin signaling by binding light-activated, phosphorylated rhodopsin and modulating downstream transducin-mediated phototransduction. This desensitization step is essential for recovery kinetics, signal fidelity, and protection of rod cells from excessive GPCR pathway activation. Visual arrestin also contributes to receptor trafficking and signaling balance within the outer segment, integrating with rhodopsin phosphorylation cycles driven by GRK1. Dysregulation or pathogenic variation in SAG is associated with inherited retinal dysfunction, making it a key target for mechanistic studies of photoreceptor stress, degeneration pathways, and visual signal transduction.

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

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

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