SIRT5 CRISPR Activation Plasmid (h): sc-416994-ACT

Human SIRT5 encodes a mitochondrial NAD+-dependent sirtuin that primarily functions as a lysine desuccinylase, demalonylase, and deglutarylase, thereby remodeling the acylation landscape of metabolic enzymes. By regulating targets in the TCA cycle, fatty acid oxidation, ketone body metabolism, and the urea cycle, SIRT5 coordinates mitochondrial energy homeostasis and redox balance under changing nutrient conditions. Altered SIRT5 activity has been associated with mitochondrial dysfunction, oxidative stress responses, and rewired intermediary metabolism observed across diverse disease-related cellular states, including cancer biology and cardiometabolic and neurodegeneration-linked phenotypes. These features make SIRT5 a useful node for studying post-translational control of mitochondrial pathways and stress-adaptive metabolism.

SIRT5 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous SIRT5 expression without altering the underlying DNA sequence.

SIRT5 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the SIRT5 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 SIRT5 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous SIRT5 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native SIRT5 locus and enabling the study of SIRT5-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of SIRT5 pathway restoration in tumor cells with silenced or reduced SIRT5 expression.

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