Orai1 CRISPR Activation Plasmid (h): sc-400901-ACT

ORAI1 encodes Orai1, the pore-forming subunit of the calcium release–activated calcium (CRAC) channel that mediates store-operated calcium entry (SOCE) following endoplasmic reticulum Ca²⁺ depletion. By enabling sustained Ca²⁺ influx, Orai1 regulates downstream Ca²⁺-dependent transcriptional programs such as NFAT signaling, shaping lymphocyte activation, cytokine production, and broader immune cell effector functions. ORAI1 activity also influences proliferation, migration, and secretion in multiple cell types through Ca²⁺-sensitive pathways and ion homeostasis networks. Dysregulated ORAI1/SOCE signaling has been linked to immune dysfunction phenotypes and inflammatory pathobiology, supporting its use as a mechanistic node in cellular signaling studies.

Orai1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous ORAI1 expression without altering the underlying DNA sequence.

Orai1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the ORAI1 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 ORAI1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Orai1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native ORAI1 locus and enabling the study of Orai1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Orai1 pathway restoration in tumor cells with silenced or reduced ORAI1 expression.

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