Mitofilin CRISPR Activation Plasmid (h): sc-403617-ACT

Human IMMT encodes mitofilin, an essential inner mitochondrial membrane scaffold protein and core component of the MICOS (mitochondrial contact site and cristae organizing system) complex. Mitofilin coordinates crista junction formation, stabilizes respiratory chain organization, and supports mitochondrial protein import and lipid trafficking at intermembrane contact sites, linking mitochondrial architecture to oxidative phosphorylation and metabolic homeostasis. Disruption of IMMT-dependent cristae structure is associated with altered mitochondrial dynamics, impaired bioenergetics, and susceptibility to cellular stress responses. IMMT has been investigated in the context of disorders featuring mitochondrial dysfunction, including neurodegenerative and cardiometabolic disease biology, where cristae remodeling can influence signaling and cell survival pathways.

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

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

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