TFIIB CRISPR Activation Plasmid (h): sc-401174-ACT

GTF2B encodes human transcription factor IIB (TFIIB), a core component of the RNA polymerase II preinitiation complex that helps position Pol II at promoters and supports start site selection during basal transcription. By bridging interactions among TBP/TFIID, TFIIF, and Pol II, TFIIB contributes to regulated transcriptional programs that govern cell cycle control, differentiation, and stress-responsive gene expression. Perturbation of general transcription machinery can reshape global transcriptomes and has been associated with oncogenic transcriptional states and altered proliferative capacity in multiple model systems. As a central regulator of Pol II initiation, TFIIB is routinely studied in promoter architecture, transcription factor recruitment, and chromatin-dependent control of gene expression.

TFIIB CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous GTF2B expression without altering the underlying DNA sequence.

TFIIB CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the GTF2B 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 GTF2B transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous TFIIB expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native GTF2B locus and enabling the study of TFIIB-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of TFIIB pathway restoration in tumor cells with silenced or reduced GTF2B expression.

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