DNA pol θ CRISPR Activation Plasmid (h): sc-407414-ACT

POLQ encodes DNA polymerase theta (DNA pol θ), an error-prone A-family polymerase with helicase-like activity that is central to microhomology-mediated end joining (MMEJ), also termed polymerase theta–mediated end joining (TMEJ). DNA pol θ supports repair of double-strand breaks and contributes to replication stress tolerance, influencing genome stability through alternative DNA end-joining pathways that operate when classical NHEJ or homologous recombination is compromised. POLQ activity has been linked to mutational signatures and chromosomal rearrangements associated with DNA damage responses, making it relevant to studies of genomic instability, replication-associated lesions, and pathway choice in double-strand break repair. As a result, POLQ is widely investigated in mechanisms of DNA repair, synthetic lethal interactions, and models of disease-associated DNA damage phenotypes.

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

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

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