SIRT4 CRISPR Activation Plasmid (h): sc-401187-ACT

Human SIRT4 encodes a mitochondrial sirtuin with NAD+-dependent enzymatic activity that modulates metabolic enzyme function through ADP-ribosylation and deacylation, influencing glutamine utilization, fatty acid oxidation, and insulin secretion. By regulating mitochondrial bioenergetics and anaplerotic flux, SIRT4 contributes to cellular adaptation to nutrient availability and oxidative stress. SIRT4 activity intersects with pathways controlling mitochondrial homeostasis, including redox balance and metabolic checkpoint signaling. Dysregulated SIRT4 expression has been associated with metabolic rewiring observed in cancer biology, diabetes-related phenotypes, and other disorders linked to mitochondrial dysfunction.

SIRT4 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous SIRT4 expression without altering the underlying DNA sequence.

SIRT4 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the SIRT4 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 SIRT4 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous SIRT4 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native SIRT4 locus and enabling the study of SIRT4-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of SIRT4 pathway restoration in tumor cells with silenced or reduced SIRT4 expression.

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