Nix CRISPR Activation Plasmid (m): sc-419357-ACT

Bnip3l (Nix) is a mitochondrial outer membrane protein that functions as a selective autophagy receptor linking damaged mitochondria to LC3-positive autophagosomes through its LIR motif. In mouse cells, Nix participates in mitochondrial quality control, apoptosis-associated signaling, and hypoxia-responsive pathways, integrating cues that influence oxidative metabolism and cellular survival. It is well known for roles in programmed mitophagy during erythroid maturation and for shaping mitochondrial turnover in metabolically stressed tissues. Dysregulated BNIP3L/Nix activity has been associated with altered mitochondrial homeostasis and cell fate decisions relevant to neurodegeneration, cardiac stress responses, and cancer biology in experimental models.

Nix CRISPR Activation Plasmid (m) provides a targeted, non-destructive approach to upregulating endogenous Bnip3l expression without altering the underlying DNA sequence.

Nix CRISPR Activation Plasmid (m) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the Bnip3l 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 Bnip3l transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Nix expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native Bnip3l locus and enabling the study of Nix-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Nix pathway restoration in tumor cells with silenced or reduced Bnip3l expression.

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