IDO CRISPR Activation Plasmid (h): sc-400495-ACT

Human IDO1 encodes indoleamine 2,3-dioxygenase (IDO), a heme-dependent, rate-limiting enzyme of the kynurenine pathway that catalyzes oxidative cleavage of tryptophan to N-formylkynurenine. By controlling local tryptophan availability and generating bioactive kynurenine metabolites, IDO influences cellular stress responses, immune signaling, and metabolic crosstalk within the tissue microenvironment. IDO1 expression is inducible by inflammatory cues, particularly interferon-driven pathways, and is frequently studied in contexts where metabolic regulation intersects with antigen presentation and tolerance mechanisms. Dysregulated IDO1 activity has been associated with inflammatory disease biology and tumor-immune interactions, making it a useful node for mechanistic studies of immunometabolism.

IDO CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous IDO1 expression without altering the underlying DNA sequence.

IDO CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the IDO1 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 IDO1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous IDO expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native IDO1 locus and enabling the study of IDO-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of IDO pathway restoration in tumor cells with silenced or reduced IDO1 expression.

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