CYP27A1 CRISPR Activation Plasmid (h): sc-402836-ACT

CYP27A1 encodes sterol 27-hydroxylase, a mitochondrial cytochrome P450 enzyme that catalyzes oxidative steps in bile acid biosynthesis from cholesterol and contributes to sterol and oxysterol metabolism. By generating intermediates such as 27-hydroxycholesterol, CYP27A1 influences cholesterol homeostasis, lipid transport, and signaling pathways linked to mitochondrial function and metabolic regulation. Perturbation of CYP27A1 activity is associated with altered bile acid composition and sterol accumulation, and is implicated in the inherited lipid storage disorder cerebrotendinous xanthomatosis (CTX). These functions make CYP27A1 a useful target for studying mitochondrial steroid oxidation, metabolic rewiring, and sterol-driven transcriptional responses in human cells.

CYP27A1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous CYP27A1 expression without altering the underlying DNA sequence.

CYP27A1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the CYP27A1 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 CYP27A1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous CYP27A1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native CYP27A1 locus and enabling the study of CYP27A1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of CYP27A1 pathway restoration in tumor cells with silenced or reduced CYP27A1 expression.

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