P5CR CRISPR Activation Plasmid (h): sc-410971-ACT

Human PYCR1 encodes pyrroline-5-carboxylate reductase 1 (P5CR), a mitochondrial enzyme that catalyzes the NAD(P)H-dependent reduction of pyrroline-5-carboxylate to proline in the terminal step of proline biosynthesis. Through coupling to glutamate and ornithine metabolism, PYCR1 supports redox homeostasis, amino acid availability, and mitochondrial function, with downstream effects on proteostasis and extracellular matrix biology. Altered PYCR1 activity has been associated with connective tissue phenotypes and metabolic stress responses, and dysregulation of proline metabolism is frequently examined in contexts such as oxidative stress signaling, collagen-rich microenvironments, and proliferative remodeling. These features make PYCR1 a useful node for investigating how mitochondrial metabolism and redox balance shape cell state and stress adaptation.

P5CR CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous PYCR1 expression without altering the underlying DNA sequence.

P5CR CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the PYCR1 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 PYCR1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous P5CR expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native PYCR1 locus and enabling the study of P5CR-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of P5CR pathway restoration in tumor cells with silenced or reduced PYCR1 expression.

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