Pol II CRISPR Activation Plasmid (h): sc-400053-ACT

POLR2A encodes the largest subunit of human RNA polymerase II (Pol II), the core enzyme that synthesizes mRNA and many noncoding RNAs and integrates promoter-proximal pausing, elongation, and co-transcriptional RNA processing. Pol II function is coordinated through the transcription cycle with phosphorylation of the C-terminal domain, coupling transcription to splicing, 5′ capping, and 3′ end formation, and linking gene expression programs to chromatin regulation and DNA damage responses. As a central node in transcriptional control, POLR2A is frequently leveraged as a reference for global transcriptional output and cellular stress adaptation. Altered Pol II activity and transcriptional dysregulation are broadly associated with oncogenic transcriptional states and neurodevelopmental phenotypes, making POLR2A relevant for mechanistic studies of gene regulation.

Pol II CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous POLR2A expression without altering the underlying DNA sequence.

Pol II CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the POLR2A 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 POLR2A transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Pol II expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native POLR2A locus and enabling the study of Pol II-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Pol II pathway restoration in tumor cells with silenced or reduced POLR2A expression.

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