Strumpellin CRISPR Activation Plasmid (h): sc-402808-ACT

WASHC5 encodes Strumpellin, an essential subunit of the WASH complex that regulates Arp2/3-dependent actin nucleation on endosomes to control membrane remodeling, endosomal tubulation, and receptor cargo sorting. Through its role in endosome-to-plasma membrane recycling and retromer-associated trafficking, Strumpellin influences signaling receptor availability, cytoskeletal organization, and cell motility. Disruption of WASHC5-dependent endosomal dynamics has been linked to neurodegenerative phenotypes, including hereditary spastic paraplegia, highlighting its relevance to neuronal maintenance and axon integrity. Strumpellin function is therefore of interest for studying endolysosomal homeostasis, actin-driven membrane dynamics, and trafficking-dependent regulation of signaling pathways.

Strumpellin CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous WASHC5 expression without altering the underlying DNA sequence.

Strumpellin CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the WASHC5 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 WASHC5 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Strumpellin expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native WASHC5 locus and enabling the study of Strumpellin-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Strumpellin pathway restoration in tumor cells with silenced or reduced WASHC5 expression.

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