NEDL2 CRISPR Activation Plasmid (h): sc-410607-ACT

Human HECW2 encodes the HECT-type E3 ubiquitin ligase NEDL2, a regulator of protein stability and signaling output through ubiquitin-mediated proteasomal turnover. NEDL2 participates in cellular quality-control and pathway modulation by targeting specific substrates for ubiquitination, thereby influencing processes such as cell-cycle control, stress responses, and transcriptional programs. Reported functional links place HECW2 within ubiquitin-dependent regulation of developmental and neuronal pathways, where altered expression or variant alleles have been associated with neurodevelopmental phenotypes. As a consequence, HECW2/NEDL2 is frequently studied in the context of proteostasis, signaling crosstalk, and genotype–phenotype relationships in human cell models.

NEDL2 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous HECW2 expression without altering the underlying DNA sequence.

NEDL2 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the HECW2 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 HECW2 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous NEDL2 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native HECW2 locus and enabling the study of NEDL2-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of NEDL2 pathway restoration in tumor cells with silenced or reduced HECW2 expression.

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