NSF CRISPR Activation Plasmid (h): sc-402258-ACT

NSF (N-ethylmaleimide–sensitive factor) encodes an AAA+ ATPase that disassembles cis-SNARE complexes after membrane fusion, regenerating SNAREs for repeated rounds of vesicular transport. This activity is central to intracellular trafficking, including exocytosis, endocytosis, and synaptic vesicle cycling, and supports organelle dynamics across the secretory and endolysosomal pathways. By controlling vesicle docking and fusion efficiency, NSF influences neurotransmission, secretion, and membrane protein turnover, processes frequently perturbed in neurological and other disorders where proteostasis and trafficking are disrupted. Dysregulated vesicle fusion machinery can alter cell signaling and stress responses, making NSF a relevant node for mechanistic studies of membrane trafficking phenotypes.

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

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

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