FAM21C CRISPR Activation Plasmid (h): sc-404093-ACT

WASHC2C encodes FAM21C, a component of the WASH complex that coordinates actin polymerization on endosomes to control membrane remodeling and cargo sorting. Through regulation of retromer-associated trafficking, FAM21C helps govern receptor recycling, endosome-to-Golgi transport, and spatial organization of signaling receptors at the cell surface. Perturbation of WASH pathway activity can reshape cytoskeletal dynamics, vesicular routing, and downstream signaling networks that influence proliferation, migration, and differentiation. Consequently, altered endosomal trafficking programs linked to WASH complex function are relevant to mechanisms studied in cancer biology, neurobiology, and immune cell regulation.

FAM21C CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous WASHC2C expression without altering the underlying DNA sequence.

FAM21C CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the WASHC2C 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 WASHC2C transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous FAM21C expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native WASHC2C locus and enabling the study of FAM21C-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of FAM21C pathway restoration in tumor cells with silenced or reduced WASHC2C expression.

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