Acid Ceramidase CRISPR Activation Plasmid (m): sc-419216-ACT

Mouse Asah1 encodes acid ceramidase, a lysosomal hydrolase that deacylates ceramide to generate sphingosine and free fatty acids, shaping the balance between sphingolipid storage and turnover. By regulating ceramide and sphingosine pools, Acid Ceramidase influences lysosome-dependent membrane trafficking, autophagy, and stress-responsive signaling networks linked to cell survival and inflammatory responses. Disruption of ASAH1 activity is associated with sphingolipid accumulation and lysosomal dysfunction, connecting this pathway to neurodegenerative and systemic metabolic phenotypes. Asah1 is therefore a key node for studying ceramide-centric lipid signaling and lysosomal homeostasis in mouse cellular and in vivo models.

Acid Ceramidase CRISPR Activation Plasmid (m) provides a targeted, non-destructive approach to upregulating endogenous Asah1 expression without altering the underlying DNA sequence.

Acid Ceramidase CRISPR Activation Plasmid (m) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the Asah1 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 Asah1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Acid Ceramidase expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native Asah1 locus and enabling the study of Acid Ceramidase-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Acid Ceramidase pathway restoration in tumor cells with silenced or reduced Asah1 expression.

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