GFRP CRISPR Activation Plasmid (h): sc-405353-ACT

Human GCHFR encodes GTP cyclohydrolase I feedback regulatory protein (GFRP), a key allosteric modulator of GTP cyclohydrolase I (GCH1) that tunes de novo tetrahydrobiopterin (BH4) biosynthesis. By integrating signals from BH4 and phenylalanine, GFRP helps maintain cofactor homeostasis that supports aromatic amino acid hydroxylation and nitric oxide synthase activity, influencing neurotransmitter production and redox-sensitive signaling. This regulation connects GCHFR to metabolic control of NO-dependent vascular and immune processes and to monoamine biosynthetic pathways in the nervous system. Dysregulated BH4 availability and altered GCH1–GFRP control have been associated with phenotypes relevant to pain signaling, endothelial dysfunction, and neurotransmitter imbalance, making GCHFR a useful node for pathway-focused studies.

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

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

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