RyR-3 CRISPR Activation Plasmid (h): sc-402031-ACT

Human RYR3 encodes ryanodine receptor 3 (RyR-3), a large endoplasmic/sarcoplasmic reticulum Ca²⁺ release channel that amplifies intracellular calcium signals in response to upstream second messengers. RyR-3 contributes to excitation–contraction coupling–adjacent calcium dynamics, calcium-induced calcium release, and broader Ca²⁺-dependent processes such as secretion, synaptic plasticity, and transcriptional programs controlled by CaMK and calcineurin/NFAT signaling. By shaping cytosolic and organellar Ca²⁺ homeostasis, RYR3 influences mitochondrial coupling, membrane excitability, and stress responses. Altered ryanodine receptor–mediated calcium handling has been implicated in neurological and muscular phenotypes and is studied in the context of arrhythmogenic and neurodevelopmental disease mechanisms without implying clinical outcomes.

RyR-3 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous RYR3 expression without altering the underlying DNA sequence.

RyR-3 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the RYR3 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 RYR3 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous RyR-3 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native RYR3 locus and enabling the study of RyR-3-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of RyR-3 pathway restoration in tumor cells with silenced or reduced RYR3 expression.

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