AKR1D1 CRISPR Activation Plasmid (h): sc-404897-ACT

AKR1D1 encodes steroid 5β-reductase, an aldo-keto reductase that catalyzes an essential step in bile acid biosynthesis by converting Δ4-3-ketosteroids to 5β-reduced intermediates. This activity supports hepatic cholesterol catabolism, bile acid homeostasis, and downstream metabolic signaling through bile acid–responsive nuclear receptors such as FXR, linking AKR1D1 to broader lipid and glucose regulatory pathways. Altered AKR1D1 expression or function can perturb bile acid composition and steroid metabolism, with relevance to cholestatic phenotypes and metabolic liver dysfunction. As a liver-enriched enzyme, AKR1D1 is frequently studied in hepatocyte models to define how bile acid flux and steroid clearance influence inflammatory and metabolic stress responses.

AKR1D1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous AKR1D1 expression without altering the underlying DNA sequence.

AKR1D1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the AKR1D1 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 AKR1D1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous AKR1D1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native AKR1D1 locus and enabling the study of AKR1D1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of AKR1D1 pathway restoration in tumor cells with silenced or reduced AKR1D1 expression.

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