OTC CRISPR Activation Plasmid (h): sc-402611-ACT

Ornithine transcarbamylase (OTC) is a mitochondrial enzyme that catalyzes the conversion of ornithine and carbamoyl phosphate to citrulline, a core step in the hepatic urea cycle responsible for ammonia detoxification. By regulating nitrogen disposal, OTC activity influences cellular nitrogen balance, mitochondrial metabolism, and amino acid homeostasis, with downstream effects on oxidative stress and bioenergetic state. Dysregulation of OTC is linked to hyperammonemia and inborn errors of metabolism, and altered urea cycle flux has been studied in contexts of metabolic rewiring and mitochondrial dysfunction. Human OTC therefore serves as a useful node for dissecting urea cycle control, nitrogen handling, and mitochondria-associated stress responses in hepatocyte and liver-relevant model systems.

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

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

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