RPB2 CRISPR Activation Plasmid (h): sc-402591-ACT

POLR2B encodes RPB2, the second-largest catalytic subunit of RNA polymerase II, which synthesizes mRNA and many noncoding RNAs essential for eukaryotic gene expression. RPB2 contributes to promoter opening, nucleotide incorporation, and transcriptional processivity, coupling transcription to co-transcriptional RNA processing and chromatin regulation. As a core component of the basal transcription machinery, POLR2B supports pathways governing cell cycle progression, differentiation, and stress responses through global control of transcriptional output. Dysregulation of RNA polymerase II function and transcriptional homeostasis is broadly relevant to proliferative and neurodevelopmental disease biology, where altered gene expression programs are a central mechanism.

POLR2B CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous POLR2B expression without altering the underlying DNA sequence.

POLR2B CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the POLR2B 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 POLR2B transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous POLR2B expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native POLR2B locus and enabling the study of POLR2B-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of POLR2B pathway restoration in tumor cells with silenced or reduced POLR2B expression.

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