EF-Tu CRISPR Activation Plasmid (h): sc-404257-ACT

TUFM encodes the human mitochondrial elongation factor Tu (EF-Tu), a core component of the mitochondrial translation machinery that delivers aminoacyl-tRNAs to the mitoribosome in a GTP-dependent manner. By supporting synthesis of oxidative phosphorylation subunits encoded by mtDNA, EF-Tu links mitochondrial protein production to respiratory chain assembly, bioenergetic flux, and proteostasis programs such as the mitochondrial unfolded protein response. Disruption of TUFM activity can perturb electron transport chain function, elevate oxidative stress, and alter mitophagy signaling, connecting EF-Tu biology to mechanisms broadly relevant to neuromuscular and neurodevelopmental disease research. As a result, TUFM is frequently studied in contexts involving mitochondrial dysfunction, metabolic remodeling, and stress adaptation.

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

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

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