Keap1 CRISPR Activation Plasmid (h): sc-400190-ACT

Human KEAP1 encodes Kelch-like ECH-associated protein 1 (Keap1), a cytosolic adaptor for a CUL3-based E3 ubiquitin ligase complex that regulates proteasomal turnover of NRF2 (NFE2L2). By sensing electrophilic and oxidative stress through reactive cysteine residues, Keap1 modulates NRF2 nuclear accumulation and transcription of antioxidant, detoxification, and metabolic response genes. This KEAP1–NRF2 axis integrates redox homeostasis with proteostasis and inflammation-related signaling, influencing cellular susceptibility to xenobiotics and mitochondrial stress. Dysregulation of KEAP1 function and altered NRF2 activity are frequently studied in cancer biology and other disorders characterized by oxidative stress and metabolic reprogramming.

Keap1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous KEAP1 expression without altering the underlying DNA sequence.

Keap1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the KEAP1 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 KEAP1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Keap1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native KEAP1 locus and enabling the study of Keap1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Keap1 pathway restoration in tumor cells with silenced or reduced KEAP1 expression.

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