INSIG-1 CRISPR Activation Plasmid (h): sc-401381-ACT

INSIG1 encodes insulin induced gene 1 (INSIG-1), an ER membrane protein that functions as a sterol sensor and central negative regulator of lipid biosynthesis. INSIG-1 binds SCAP to retain the SCAP–SREBP complex in the ER under sterol-replete conditions, limiting SREBP processing and downstream transcriptional programs controlling cholesterol and fatty acid synthesis. It also interacts with sterol-accelerated degradation pathways by promoting ubiquitination of HMG-CoA reductase, thereby linking sterol availability to mevalonate pathway flux. Dysregulated INSIG1–SREBP signaling is implicated in metabolic phenotypes and contexts where altered lipogenesis supports cell growth and stress adaptation, making it relevant to studies of lipid homeostasis, ER function, and metabolic disease mechanisms.

INSIG-1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous INSIG1 expression without altering the underlying DNA sequence.

INSIG-1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the INSIG1 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 INSIG1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous INSIG-1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native INSIG1 locus and enabling the study of INSIG-1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of INSIG-1 pathway restoration in tumor cells with silenced or reduced INSIG1 expression.

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