Rb CRISPR Activation Plasmid (h): sc-400116-ACT

Human RB1 encodes the retinoblastoma protein (Rb), a central tumor suppressor that governs the G1/S checkpoint by binding E2F transcription factors and coordinating cell-cycle gene expression. Rb activity is regulated by cyclin D–CDK4/6 and cyclin E–CDK2 phosphorylation, linking mitogenic signaling to DNA replication entry, chromatin remodeling, and differentiation programs. Beyond cell-cycle control, RB1 influences genome stability, senescence, and transcriptional repression through interactions with histone modifiers and SWI/SNF complexes. Disruption of RB1 pathway signaling is broadly associated with deregulated proliferation and is frequently implicated in cancers and other proliferative disorders, making RB1 a key node for mechanistic studies of cell-cycle control.

Rb CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous RB1 expression without altering the underlying DNA sequence.

Rb CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the RB1 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 RB1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Rb expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native RB1 locus and enabling the study of Rb-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Rb pathway restoration in tumor cells with silenced or reduced RB1 expression.

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