Ku-86 CRISPR Activation Plasmid (h): sc-400549-ACT

XRCC5 encodes Ku-86 (Ku80), a core component of the Ku70/Ku80 heterodimer that recognizes DNA double-strand breaks and initiates classical non-homologous end joining (c-NHEJ). Ku-86 recruits and coordinates DNA-PKcs and downstream repair factors, integrating DNA damage sensing with end processing and ligation, and also contributes to V(D)J recombination and telomere maintenance. Through these roles, XRCC5 influences genome stability, cell-cycle checkpoint signaling, and cellular responses to genotoxic stress. Dysregulation of Ku-dependent repair and DNA-PK activity is frequently studied in contexts of chromosomal instability and altered DNA repair capacity observed across diverse disease-relevant models.

Ku-86 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous XRCC5 expression without altering the underlying DNA sequence.

Ku-86 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the XRCC5 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 XRCC5 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Ku-86 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native XRCC5 locus and enabling the study of Ku-86-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Ku-86 pathway restoration in tumor cells with silenced or reduced XRCC5 expression.

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