RNF8 CRISPR Activation Plasmid (h): sc-401909-ACT

RNF8 encodes an E3 ubiquitin ligase that functions as an early signaling amplifier in the DNA damage response, catalyzing ubiquitination at sites of DNA double-strand breaks to coordinate recruitment of repair factors. Through interactions with MDC1 and phosphorylation-dependent docking at damaged chromatin, RNF8 promotes ubiquitin-dependent assembly of downstream mediators such as 53BP1 and BRCA1, influencing pathway choice between non-homologous end joining and homologous recombination. RNF8 activity also links genome surveillance to cell-cycle checkpoint control and chromatin remodeling, helping maintain genomic stability under replication stress. Dysregulated RNF8 signaling has been associated with altered DNA repair capacity and genomic instability phenotypes relevant to cancer biology and sensitivity to genotoxic stressors.

RNF8 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous RNF8 expression without altering the underlying DNA sequence.

RNF8 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the RNF8 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 RNF8 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous RNF8 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native RNF8 locus and enabling the study of RNF8-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of RNF8 pathway restoration in tumor cells with silenced or reduced RNF8 expression.

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