IRP-1 CRISPR Activation Plasmid (h): sc-401817-ACT

Human ACO1 encodes iron regulatory protein 1 (IRP-1), a bifunctional cytosolic factor that interconverts between an enzymatically active aconitase form and an RNA-binding regulator depending on the status of its [4Fe-4S] cluster. As IRP-1, it binds iron-responsive elements (IREs) in target mRNAs to coordinate iron uptake, storage, and utilization by modulating translation and mRNA stability, integrating iron-sulfur cluster biogenesis with cellular redox and metabolic programs. Through this post-transcriptional control network, ACO1 helps maintain iron homeostasis and influences pathways linked to mitochondrial function, oxidative stress responses, and heme and iron-sulfur protein assembly. Dysregulated IRP-1/IRE signaling and altered ACO1 activity are frequently studied in contexts of iron-loading or iron-restricted states, anemia-related biology, and neurodegeneration- and cancer-associated metabolic remodeling.

IRP-1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous ACO1 expression without altering the underlying DNA sequence.

IRP-1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the ACO1 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 ACO1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous IRP-1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native ACO1 locus and enabling the study of IRP-1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of IRP-1 pathway restoration in tumor cells with silenced or reduced ACO1 expression.

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