URE-B1 CRISPR Activation Plasmid (h): sc-404890-ACT

HUWE1 encodes URE-B1, a HECT-type E3 ubiquitin ligase that regulates protein homeostasis by catalyzing ubiquitin transfer to diverse substrates involved in cell-cycle progression, DNA damage signaling, apoptosis, and stress responses. Through control of ubiquitin-dependent turnover, HUWE1 influences pathways linked to chromatin dynamics, MYC- and p53-associated networks, and mitochondrial and proteotoxic stress checkpoints. Altered HUWE1 activity or expression has been associated with dysregulated proliferation and genome integrity, and genetic variation in HUWE1 has been reported in neurodevelopmental and cancer-related contexts. As a central node in ubiquitin signaling, URE-B1 is frequently studied to dissect substrate-specific ubiquitination and pathway cross-talk in human cells.

URE-B1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous HUWE1 expression without altering the underlying DNA sequence.

URE-B1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the HUWE1 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 HUWE1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous URE-B1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native HUWE1 locus and enabling the study of URE-B1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of URE-B1 pathway restoration in tumor cells with silenced or reduced HUWE1 expression.

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