Bad CRISPR Activation Plasmid (h): sc-400419-ACT

Human BAD (BCL2 associated agonist of cell death) encodes Bad, a pro-apoptotic BH3-only member of the BCL-2 family that integrates survival and death cues at the mitochondria. Bad promotes mitochondrial outer membrane permeabilization by binding and neutralizing anti-apoptotic proteins such as BCL-2 and BCL-XL, thereby facilitating BAX/BAK activation and caspase-dependent apoptosis. Its activity is tightly regulated by phosphorylation and sequestration downstream of PI3K/AKT and MAPK signaling, linking growth factor signaling to intrinsic apoptosis and metabolic control. Dysregulated BAD signaling has been implicated in altered apoptotic thresholds relevant to cancer biology, neurodegeneration, and stress responses.

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

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

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