RPA194 CRISPR Activation Plasmid (h): sc-400816-ACT

POLR1A encodes RPA194, the largest catalytic subunit of RNA polymerase I that drives ribosomal RNA (47S pre-rRNA) synthesis in the nucleolus, a rate-limiting step for ribosome biogenesis and proteome capacity. RPA194 function integrates with nucleolar organization, rDNA transcription control, and cellular growth programs, coupling nutrient and stress signals to translational output. Disruption of Pol I transcription can elicit nucleolar stress and engage p53-dependent and p53-independent surveillance pathways, linking POLR1A regulation to genome stability and cell-cycle control. Aberrant ribosome biogenesis and altered Pol I activity are associated with proliferative phenotypes and disease-relevant states where protein synthesis demand and nucleolar function are remodeled.

RPA194 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous POLR1A expression without altering the underlying DNA sequence.

RPA194 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the POLR1A 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 POLR1A transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous RPA194 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native POLR1A locus and enabling the study of RPA194-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of RPA194 pathway restoration in tumor cells with silenced or reduced POLR1A expression.

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