ASM CRISPR Activation Plasmid (h): sc-401347-ACT

Human SMPD1 encodes acid sphingomyelinase (ASM), a lysosomal hydrolase that cleaves sphingomyelin to generate ceramide and phosphorylcholine, thereby regulating sphingolipid homeostasis. ASM activity influences membrane microdomain organization, endolysosomal trafficking, and stress-responsive signaling pathways linked to ceramide-dependent apoptosis, autophagy, and inflammatory responses. Perturbation of SMPD1 alters lysosomal function and lipid storage dynamics, with well-established relevance to sphingolipid storage disorders and broader mechanisms of neurodegeneration and immune dysregulation. As a central node in sphingomyelin–ceramide metabolism, SMPD1 is commonly studied for its impact on cellular stress adaptation and membrane-associated signaling.

ASM CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous SMPD1 expression without altering the underlying DNA sequence.

ASM CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the SMPD1 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 SMPD1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous ASM expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native SMPD1 locus and enabling the study of ASM-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of ASM pathway restoration in tumor cells with silenced or reduced SMPD1 expression.

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