RPE65 CRISPR Activation Plasmid (h): sc-401489-ACT

RPE65 encodes a retinoid isomerohydrolase that is essential for the visual cycle in retinal pigment epithelium, catalyzing the conversion of all-trans-retinyl esters to 11-cis-retinol to sustain rhodopsin regeneration. This activity supports phototransduction by maintaining chromophore availability and links retinoid metabolism to cellular processes including retinol transport, esterification, and oxidative stress responses in the outer retina. Altered RPE65 expression or function is associated with impaired chromophore recycling and photoreceptor dysfunction, making it a key node for studying retinal homeostasis. In vitro models leveraging RPE65 modulation are used to interrogate transcriptional regulation of the visual cycle and downstream signaling changes relevant to retinal degeneration phenotypes.

RPE65 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous RPE65 expression without altering the underlying DNA sequence.

RPE65 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the RPE65 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 RPE65 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous RPE65 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native RPE65 locus and enabling the study of RPE65-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of RPE65 pathway restoration in tumor cells with silenced or reduced RPE65 expression.

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