mtTFA CRISPR Activation Plasmid (m): sc-423355-ACT

Tfam encodes mitochondrial transcription factor A (mtTFA), a high-mobility group protein that binds mitochondrial DNA to package nucleoids and regulate mitochondrial genome transcription and replication. mtTFA is a central determinant of mtDNA copy number and mitochondrial gene expression, thereby influencing oxidative phosphorylation, ATP production, and reactive oxygen species homeostasis. Through these roles it integrates with mitochondrial biogenesis programs and nuclear–mitochondrial communication pathways that shape cellular metabolism and stress responses. Altered TFAM activity and mtDNA maintenance are frequently investigated in models of neuromuscular dysfunction, neurodegeneration, cardiometabolic phenotypes, and inflammation driven by mitochondrial stress signaling.

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

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

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