DNA pol ζ CRISPR Activation Plasmid (h): sc-402579-ACT

REV3L encodes the catalytic subunit of DNA polymerase ζ (pol ζ), a specialized translesion synthesis polymerase that extends from nucleotides inserted opposite DNA lesions to support replication fork progression under genotoxic stress. In coordination with REV7 (MAD2L2) and ubiquitin signaling at stalled forks, pol ζ functions within DNA damage tolerance pathways that interface with replication stress responses and post-replication repair. Because pol ζ is intrinsically error-prone, REV3L activity influences mutational burden and genome stability, linking its regulation to processes such as carcinogenesis and resistance to DNA-damaging exposures in model systems. Altered REV3L function also impacts cell-cycle progression and checkpoint signaling by modulating the outcome of lesion bypass versus fork collapse.

DNA pol ζ CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous REV3L expression without altering the underlying DNA sequence.

DNA pol ζ CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the REV3L 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 REV3L transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous DNA pol ζ expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native REV3L locus and enabling the study of DNA pol ζ-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of DNA pol ζ pathway restoration in tumor cells with silenced or reduced REV3L expression.

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