RyR CRISPR Activation Plasmid (h): sc-401470-ACT

Human RYR1 encodes the ryanodine receptor (RyR1), a tetrameric Ca²⁺ release channel of the sarcoplasmic/endoplasmic reticulum that couples membrane depolarization to rapid intracellular calcium mobilization. RyR1-driven Ca²⁺ transients regulate excitation–contraction coupling, calcium-induced calcium release, and downstream signaling pathways that shape mitochondrial metabolism, cytoskeletal dynamics, and transcriptional responses to calcium. RYR1 activity is tightly controlled by luminal Ca²⁺, redox state, and accessory proteins such as FKBP12, calmodulin, and triadin/junctin complexes. Dysregulated RyR1 channel gating and aberrant Ca²⁺ homeostasis are linked to skeletal muscle pathophysiology and are frequently studied in the context of malignant hyperthermia susceptibility and congenital myopathies.

RyR-1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous RYR1 expression without altering the underlying DNA sequence.

RyR-1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the RYR1 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 RYR1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous RyR-1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native RYR1 locus and enabling the study of RyR-1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of RyR-1 pathway restoration in tumor cells with silenced or reduced RYR1 expression.

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