Rad54 CRISPR Activation Plasmid (h): sc-401750-ACT

RAD54L encodes the human Rad54 DNA-dependent ATPase, a SWI2/SNF2-family chromatin remodeling factor that partners with RAD51 to promote homology search, D-loop formation, and branch migration during homologous recombination. Rad54 functions in the DNA damage response to resolve replication-associated lesions and double-strand breaks, supporting genome stability through S and G2 phase repair pathways. Altered RAD54L activity can perturb recombination fidelity and contribute to mutational burden and chromosomal rearrangements observed in cancer biology and hereditary genome instability contexts. RAD54L is therefore widely studied in mechanisms of replication stress, recombination-mediated repair, and pathway choice between homologous recombination and end-joining.

Rad54 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous RAD54L expression without altering the underlying DNA sequence.

Rad54 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the RAD54L 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 RAD54L transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Rad54 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native RAD54L locus and enabling the study of Rad54-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Rad54 pathway restoration in tumor cells with silenced or reduced RAD54L expression.

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