UBA2 CRISPR Activation Plasmid (h): sc-404495-ACT

UBA2 encodes the SUMO-activating enzyme subunit 2, which heterodimerizes with SAE1 to form the E1 enzyme that initiates SUMOylation by adenylating SUMO and transferring it to the E2 conjugating enzyme UBC9. This pathway regulates protein stability, subcellular localization, and activity across key processes including DNA damage signaling, chromatin organization, transcriptional control, and cell-cycle progression. UBA2-dependent SUMOylation influences stress responses and proteostasis through coordination with ubiquitin-like modification networks. Dysregulated SUMO pathway activity, including altered UBA2 function or expression, has been implicated in oncogenic transcription programs and genome maintenance defects that are relevant to cancer biology and other proliferative or stress-associated phenotypes.

UBA2 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous UBA2 expression without altering the underlying DNA sequence.

UBA2 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the UBA2 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 UBA2 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous UBA2 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native UBA2 locus and enabling the study of UBA2-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of UBA2 pathway restoration in tumor cells with silenced or reduced UBA2 expression.

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