IRE1α CRISPR Activation Plasmid (h): sc-400576-ACT

ERN1 encodes inositol-requiring enzyme 1 alpha (IRE1α), an ER-resident transmembrane kinase/endoribonuclease that functions as a core sensor of unfolded protein load and initiates the unfolded protein response (UPR). Upon ER stress, IRE1α oligomerizes and activates its RNase activity to splice XBP1 mRNA and promote adaptive transcriptional programs, while also engaging regulated IRE1-dependent decay (RIDD) to modulate mRNA homeostasis. Through crosstalk with PERK and ATF6 signaling, ERN1 coordinates proteostasis, lipid metabolism, and inflammatory signaling, influencing cell fate decisions between adaptation and apoptosis. Dysregulated IRE1α–XBP1 axis activity has been implicated in contexts such as tumor cell survival under stress, metabolic dysfunction, and neurodegeneration, making ERN1 a widely used node for mechanistic studies of ER stress biology.

IRE1α CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous ERN1 expression without altering the underlying DNA sequence.

IRE1α CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the ERN1 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 ERN1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous IRE1α expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native ERN1 locus and enabling the study of IRE1α-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of IRE1α pathway restoration in tumor cells with silenced or reduced ERN1 expression.

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