NEDD8 CRISPR Activation Plasmid (h): sc-417924-ACT

NEDD8 encodes the ubiquitin-like protein NEDD8, a key modifier in the neddylation pathway that covalently conjugates to cullin scaffold proteins to activate cullin–RING E3 ubiquitin ligases (CRLs). Through regulation of CRL-dependent ubiquitination, NEDD8 influences proteostasis, cell-cycle progression, DNA replication and repair, and stress-response signaling. NEDD8-dependent CRL activity shapes turnover of many short-lived regulators, linking neddylation to checkpoint control and transcriptional programs. Dysregulated neddylation and altered CRL function are frequently studied in the context of proliferative and proteotoxic phenotypes, genome instability, and pathway remodeling observed across diverse disease models.

NEDD8 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous NEDD8 expression without altering the underlying DNA sequence.

NEDD8 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the NEDD8 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 NEDD8 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous NEDD8 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native NEDD8 locus and enabling the study of NEDD8-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of NEDD8 pathway restoration in tumor cells with silenced or reduced NEDD8 expression.

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