DNA-PK_CS CRISPR Activation Plasmid (h): sc-400337-ACT

PRKDC encodes the catalytic subunit of DNA-dependent protein kinase (DNA-PKCS), a central regulator of the DNA damage response that couples DNA end recognition to repair. DNA-PKCS forms the DNA-PK holoenzyme with the Ku70/Ku80 heterodimer to coordinate non-homologous end joining (NHEJ), promoting double-strand break repair, V(D)J recombination, and maintenance of genome stability. Through phosphorylation of itself and multiple repair and signaling factors, DNA-PKCS integrates with ATM/ATR signaling, replication stress responses, and chromatin remodeling. Dysregulated PRKDC activity or expression is linked to altered radiosensitivity, immunodeficiency phenotypes, and genomic instability observed across diverse cancer and neurodegeneration research contexts.

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

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

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