IscU1/2 CRISPR Activation Plasmid (h): sc-402325-ACT

Human ISCU encodes the IscU1/2 scaffold proteins that coordinate de novo iron–sulfur (Fe–S) cluster biogenesis in mitochondria and the cytosol, supporting the maturation of Fe–S-dependent enzymes involved in oxidative phosphorylation, TCA cycle activity, and DNA maintenance. By serving as a transient assembly platform for cluster transfer to client proteins, IscU1/2 links mitochondrial metabolism to redox homeostasis and cellular stress responses. Perturbation of ISCU expression or splicing disrupts Fe–S enzyme function, with downstream effects on energy production and iron handling, and has been associated with mitochondrial myopathy and broader neuromuscular and metabolic phenotypes. These features make ISCU a relevant node for studying mitochondrial proteostasis, iron metabolism, and metabolic adaptation in human cell models.

IscU1/2 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous ISCU expression without altering the underlying DNA sequence.

IscU1/2 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the ISCU 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 ISCU transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous IscU1/2 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native ISCU locus and enabling the study of IscU1/2-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of IscU1/2 pathway restoration in tumor cells with silenced or reduced ISCU expression.

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