PCNA CRISPR Activation Plasmid (h2): sc-400037-ACT-2

Human PCNA (proliferating cell nuclear antigen) is a homotrimeric DNA sliding clamp that functions as a central scaffold for DNA polymerases and numerous repair and chromatin-modifying factors, coordinating high-processivity DNA synthesis during S phase. It integrates replication with DNA damage tolerance and repair pathways, including mismatch repair, base excision repair, nucleotide excision repair, and translesion synthesis, and its activity is regulated by post-translational modifications such as ubiquitination and SUMOylation that influence partner selection. Dysregulated PCNA expression or interaction networks are frequently associated with genomic instability and aberrant proliferation observed in cancer and other replication-stress–linked pathologies. PCNA is widely used in biomedical research as a marker of cell-cycle progression and as a mechanistic entry point to interrogate replisome architecture, replication fork dynamics, and repair pathway choice in human cells.

PCNA CRISPR Activation Plasmid (h2) provides a targeted, non-destructive approach to upregulating endogenous PCNA expression without altering the underlying DNA sequence.

PCNA CRISPR Activation Plasmid (h2) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the PCNA 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 PCNA transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous PCNA expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native PCNA locus and enabling the study of PCNA-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of PCNA pathway restoration in tumor cells with silenced or reduced PCNA expression.

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