BRCA2 CRISPR Activation Plasmid (h): sc-400700-ACT

Human BRCA2 encodes a tumor suppressor that is essential for high-fidelity repair of DNA double-strand breaks through homologous recombination by mediating RAD51 loading onto resected DNA and stabilizing replication forks during S phase. BRCA2 functions within the Fanconi anemia/BRCA network to resolve DNA interstrand crosslinks and coordinate checkpoint signaling to preserve genome integrity. Disruption or reduced BRCA2 activity increases chromosomal instability and elevates susceptibility to malignancy, making it a central gene in studies of DNA damage responses. BRCA2 status also influences cellular sensitivity to replication stress and genotoxic agents, supporting broad applications in genome maintenance and cancer biology research.

BRCA2 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous BRCA2 expression without altering the underlying DNA sequence.

BRCA2 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the BRCA2 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 BRCA2 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous BRCA2 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native BRCA2 locus and enabling the study of BRCA2-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of BRCA2 pathway restoration in tumor cells with silenced or reduced BRCA2 expression.

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