IRS-1 CRISPR Activation Plasmid (h): sc-400096-ACT

Human IRS1 encodes insulin receptor substrate 1 (IRS-1), a central adaptor protein that couples activated insulin and IGF-1 receptors to downstream PI3K–AKT–mTOR and RAS–MAPK signaling. Through tyrosine phosphorylation–dependent recruitment of SH2-domain effectors, IRS-1 regulates glucose uptake, glycogen synthesis, protein translation, cell growth, and survival, while serine/threonine phosphorylation provides key negative-feedback control. Dysregulated IRS-1 signaling and post-translational modification patterns are widely used molecular readouts in studies of insulin resistance, metabolic stress, and growth factor–driven phenotypes. Altered IRS1 expression or signaling network connectivity has also been investigated in contexts including type 2 diabetes, obesity-associated inflammation, and cancer cell metabolism.

IRS-1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous IRS1 expression without altering the underlying DNA sequence.

IRS-1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the IRS1 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 IRS1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous IRS-1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native IRS1 locus and enabling the study of IRS-1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of IRS-1 pathway restoration in tumor cells with silenced or reduced IRS1 expression.

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