rhodopsin CRISPR Activation Plasmid (h): sc-401026-ACT

Human RHO encodes rhodopsin, a light-sensitive G protein-coupled receptor embedded in rod photoreceptor outer segment discs that initiates the phototransduction cascade. Upon photon absorption, rhodopsin activates transducin to modulate cGMP signaling, controlling cyclic nucleotide–gated channel activity and downstream membrane hyperpolarization critical for dim-light vision. RHO expression and protein turnover are tightly linked to retinal homeostasis processes including chromophore regeneration in the visual cycle, trafficking to outer segments, and regulated GPCR deactivation. Dysregulation or mutation of RHO is associated with inherited retinal degenerations, making it a key target for mechanistic studies of GPCR signaling, photoreceptor stress responses, and gene regulation in retinal models.

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

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

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