CHAC1 CRISPR Activation Plasmid (h): sc-403197-ACT

CHAC1 (ChaC glutathione-specific gamma-glutamylcyclotransferase 1) is a stress-inducible enzyme that degrades glutathione, linking redox homeostasis to amino acid metabolism and cellular adaptation to oxidative and endoplasmic reticulum stress. As a downstream effector of the ATF4-driven integrated stress response, CHAC1 influences reactive oxygen species balance, protein folding capacity, and susceptibility to stress-associated cell fate programs. Altered CHAC1 expression has been reported across contexts involving metabolic stress and inflammation, including cancer biology and neurodegenerative disease models, where glutathione depletion and ferroptosis-related pathways are frequently interrogated. Modulating CHAC1 provides a tractable approach to study how glutathione turnover reshapes signaling networks, mitochondrial function, and stress-responsive transcriptional programs in human cells.

CHAC1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous CHAC1 expression without altering the underlying DNA sequence.

CHAC1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the CHAC1 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 CHAC1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous CHAC1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native CHAC1 locus and enabling the study of CHAC1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of CHAC1 pathway restoration in tumor cells with silenced or reduced CHAC1 expression.

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