Spastin CRISPR Activation Plasmid (h): sc-402225-ACT

Human SPAST encodes spastin, an AAA ATPase microtubule-severing enzyme that remodels the microtubule cytoskeleton by extracting tubulin subunits from the lattice. Spastin supports axonal maintenance, endosomal and ER trafficking, and membrane fission events, integrating with cytoskeletal dynamics that shape neurite outgrowth and intracellular transport. Through these roles, SPAST influences pathways governing organelle distribution and neuronal connectivity in long projection neurons. Dysregulation or loss of spastin activity is strongly linked to hereditary spastic paraplegia and is frequently studied in models of axonopathy and neurodegeneration-associated trafficking defects.

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

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

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