Rad1 CRISPR Activation Plasmid (h): sc-403600-ACT

Human RAD1 encodes Rad1, a core component of the RAD9A–HUS1–RAD1 (9-1-1) checkpoint clamp that is loaded onto damaged DNA to coordinate genome surveillance. Rad1 supports ATR-mediated signaling, promotes replication fork stability, and interfaces with DNA repair pathways including base excision repair and nucleotide excision repair to maintain chromosomal integrity. Through these functions, RAD1 influences cell-cycle checkpoint control, replication stress responses, and the resolution of DNA lesions that otherwise drive genomic instability. Dysregulated checkpoint and repair capacity involving the 9-1-1 complex has been linked to cancer-associated DNA damage phenotypes and sensitivity to genotoxic stress in experimental models.

Rad1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous RAD1 expression without altering the underlying DNA sequence.

Rad1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the RAD1 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 RAD1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Rad1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native RAD1 locus and enabling the study of Rad1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Rad1 pathway restoration in tumor cells with silenced or reduced RAD1 expression.

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