REDD-1 CRISPR Activation Plasmid (h): sc-401797-ACT

Human DDIT4 encodes REDD-1, a stress-inducible regulator that integrates hypoxia, DNA damage, ER stress, and nutrient deprivation signals to restrain mTORC1 activity through upstream modulation of the TSC1/2–Rheb axis. By dampening anabolic growth programs, REDD-1 influences protein synthesis, autophagy, mitochondrial metabolism, and redox homeostasis, shaping cellular adaptation to fluctuating microenvironments. Altered DDIT4/REDD-1 expression has been reported across contexts characterized by metabolic stress and dysregulated mTOR signaling, including cancer biology, neurodegeneration, and inflammatory states, where it can impact survival and stress tolerance. As a transcriptionally responsive node, REDD-1 is frequently used to probe pathway coupling between HIF-1 signaling, AMPK-dependent energy sensing, and mTOR-controlled growth outputs.

REDD-1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous DDIT4 expression without altering the underlying DNA sequence.

REDD-1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the DDIT4 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 DDIT4 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous REDD-1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native DDIT4 locus and enabling the study of REDD-1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of REDD-1 pathway restoration in tumor cells with silenced or reduced DDIT4 expression.

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