HMGCL CRISPR Activation Plasmid (h): sc-403841-ACT

Human HMGCL encodes 3-hydroxymethyl-3-methylglutaryl-CoA lyase, a mitochondrial enzyme that catalyzes the final step of ketogenesis and contributes to leucine catabolism by generating acetoacetate and acetyl-CoA. This activity supports cellular adaptation to fasting and metabolic stress by linking fatty acid–derived acetyl-CoA flux to production of ketone bodies and downstream energy homeostasis. HMGCL function interfaces with mitochondrial carbon metabolism and redox balance, influencing availability of acetyl-CoA for biosynthetic and signaling processes. Altered HMGCL activity is associated with inborn errors of metabolism affecting ketone body production and can be investigated in contexts where mitochondrial metabolic rewiring and nutrient utilization impact cellular phenotypes.

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

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

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