CTH CRISPR Activation Plasmid (m): sc-430767-ACT

Mouse Cth encodes cystathionine gamma-lyase (CTH), a pyridoxal phosphate–dependent enzyme of the transsulfuration pathway that converts cystathionine to cysteine, α-ketobutyrate, and ammonia and contributes to cellular hydrogen sulfide (H₂S) production. Through regulation of cysteine availability and redox buffering, CTH influences glutathione metabolism, oxidative stress responses, mitochondrial function, and metabolic homeostasis. Altered CTH activity has been linked to perturbations in sulfur amino acid metabolism and H₂S-dependent signaling that intersect with inflammatory pathways, vascular biology, and neurobiology. As a result, CTH is frequently studied in models of oxidative stress, metabolic dysfunction, and tissue injury to clarify how sulfur flux modulates cell state and signaling networks.

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

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

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