Rad50 CRISPR Activation Plasmid (h): sc-401617-ACT

Human RAD50 encodes Rad50, a core component of the MRN (MRE11-RAD50-NBN) complex that detects DNA double-strand breaks and coordinates DNA damage response signaling. Rad50’s ATPase and tethering activities help bridge broken DNA ends and support homologous recombination, non-homologous end joining, replication fork stability, and telomere maintenance. Through functional coupling to ATM-dependent checkpoint activation and end resection control, RAD50 influences cell-cycle arrest and genome surveillance programs. Dysregulation of MRN complex activity and RAD50-associated DNA repair defects are linked to genomic instability phenotypes that are frequently studied in cancer biology and inherited DNA repair disorders.

Rad50 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous RAD50 expression without altering the underlying DNA sequence.

Rad50 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the RAD50 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 RAD50 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Rad50 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native RAD50 locus and enabling the study of Rad50-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Rad50 pathway restoration in tumor cells with silenced or reduced RAD50 expression.

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