Aminoacylase-1 CRISPR Activation Plasmid (h): sc-408464-ACT

ACY1 encodes aminoacylase-1, a zinc-dependent cytosolic hydrolase that catalyzes deacylation of N-acyl-L-amino acids to generate free amino acids and acetate. This activity supports amino acid salvage and nitrogen metabolism and can influence intracellular pools of substrates that intersect with broader metabolic and proteostasis pathways. Aminoacylase-1 is also implicated in handling acetylated amino acid derivatives that arise from peptide turnover, linking its function to cellular homeostasis under metabolic stress. Altered ACY1 expression has been reported across multiple disease contexts, including cancer-associated metabolic reprogramming and inborn errors involving amino acid metabolism, making it a useful node for mechanistic studies.

Aminoacylase-1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous ACY1 expression without altering the underlying DNA sequence.

Aminoacylase-1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the ACY1 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 ACY1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Aminoacylase-1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native ACY1 locus and enabling the study of Aminoacylase-1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Aminoacylase-1 pathway restoration in tumor cells with silenced or reduced ACY1 expression.

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