BCDO2 CRISPR Activation Plasmid (h): sc-413515-ACT

Human BCO2 encodes beta-carotene oxygenase 2 (BCDO2), a mitochondrial carotenoid-cleaving enzyme that catalyzes oxidative cleavage of carotenoids and related apocarotenoids, thereby influencing cellular retinoid balance and antioxidant homeostasis. By limiting carotenoid accumulation in mitochondria, BCDO2 contributes to mitochondrial integrity, regulation of reactive oxygen species, and metabolic stress responses that intersect with lipid metabolism and redox signaling. Perturbation of BCO2 expression has been linked in the literature to altered carotenoid/retinoid-derived signaling and mitochondrial dysfunction, processes relevant to metabolic phenotypes and oxidative stress-associated tissue pathology. These features make BCO2 a useful target for mechanistic studies of carotenoid metabolism, mitochondrial quality control, and context-dependent transcriptional programs.

BCDO2 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous BCO2 expression without altering the underlying DNA sequence.

BCDO2 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the BCO2 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 BCO2 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous BCDO2 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native BCO2 locus and enabling the study of BCDO2-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of BCDO2 pathway restoration in tumor cells with silenced or reduced BCO2 expression.

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