UBC13 CRISPR Activation Plasmid (h): sc-417204-ACT

Human UBE2N encodes the E2 ubiquitin-conjugating enzyme UBC13, a core catalyst of Lys63-linked polyubiquitination that shapes non-proteolytic signaling outputs. In complex with UEV cofactors such as UBE2V1/UBE2V2, UBC13 supports assembly of K63 ubiquitin chains that coordinate DNA damage responses, replication stress tolerance, and innate immune signaling. This activity is central to activation of pathways including NF-κB and MAPK downstream of receptors such as TNFR and Toll-like receptors, and it influences protein complex dynamics rather than proteasomal turnover. Dysregulated UBE2N/UBC13-dependent ubiquitin signaling has been implicated in altered inflammatory states, genome stability defects, and oncogenic pathway rewiring, making it relevant for mechanistic studies in cancer biology and immunology.

UBC13 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous UBE2N expression without altering the underlying DNA sequence.

UBC13 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the UBE2N 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 UBE2N transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous UBC13 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native UBE2N locus and enabling the study of UBC13-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of UBC13 pathway restoration in tumor cells with silenced or reduced UBE2N expression.

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