DNA pol α CRISPR Activation Plasmid (h): sc-402762-ACT

POLA1 encodes the catalytic subunit of human DNA polymerase alpha (DNA pol α), a primase-associated polymerase essential for initiating chromosomal DNA replication by extending RNA–DNA primers synthesized at replication origins and on the lagging strand. This activity positions POLA1 at the core of the replication initiation program, coordinating with origin licensing and replisome assembly to support S-phase progression and genome stability. Perturbation of DNA pol α function can promote replication stress, altered fork dynamics, and downstream DNA damage responses that intersect with checkpoint signaling and chromatin maintenance pathways. POLA1 has been implicated in disorders linked to defective nucleic acid metabolism and immune signaling, and it is frequently studied in the context of proliferative control and replication-associated genome instability.

DNA pol α CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous POLA1 expression without altering the underlying DNA sequence.

DNA pol α CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the POLA1 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 POLA1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous DNA pol α expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native POLA1 locus and enabling the study of DNA pol α-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of DNA pol α pathway restoration in tumor cells with silenced or reduced POLA1 expression.

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