ASAHL CRISPR Activation Plasmid (h): sc-407543-ACT

Human NAAA encodes N-acylethanolamine acid amidase (ASAHL), a lysosomal enzyme that hydrolyzes fatty acid ethanolamides such as palmitoylethanolamide to their corresponding fatty acids and ethanolamine. By controlling intracellular pools of bioactive N-acylethanolamines, ASAHL influences lipid signaling networks that intersect with inflammatory regulation, cellular stress responses, and immune cell function. NAAA/ASAHL activity is commonly studied in the context of neuroinflammation, pain biology, and metabolic inflammation, where altered lipid mediator turnover can shift pathway tone without changing upstream biosynthesis. Its lysosome-associated role also makes it relevant for investigating organelle homeostasis and lipid catabolism in disease-relevant cellular models.

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

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

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