RNF168 CRISPR Activation Plasmid (h): sc-402174-ACT

RNF168 encodes an E3 ubiquitin ligase that amplifies DNA damage signaling at chromatin by ubiquitinating histone H2A-type substrates at sites of double-strand breaks. This ubiquitin-dependent cascade promotes recruitment and retention of key repair factors, supporting coordinated DNA damage response pathways including ATM signaling, checkpoint control, and repair pathway choice. RNF168 activity is tightly linked to genome stability, where altered function can disrupt chromatin-based damage signaling and compromise faithful repair. Dysregulation of RNF168-associated ubiquitin signaling has been studied in the context of inherited DNA repair defects and tumor biology, making it relevant for mechanistic investigations of genomic maintenance.

RNF168 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous RNF168 expression without altering the underlying DNA sequence.

RNF168 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the RNF168 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 RNF168 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous RNF168 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native RNF168 locus and enabling the study of RNF168-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of RNF168 pathway restoration in tumor cells with silenced or reduced RNF168 expression.

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