RANK CRISPR Activation Plasmid (h): sc-400559-ACT

TNFRSF11A encodes RANK, a TNF receptor superfamily member that functions as a key signaling node in RANKL-driven control of osteoclast differentiation, activation, and survival. Upon ligand engagement, RANK recruits adaptor proteins such as TRAFs to initiate canonical and noncanonical NF-κB signaling, MAPK cascades, and AP-1/NFATc1-dependent transcriptional programs. These pathways integrate cues from the bone microenvironment and immune system, linking RANK activity to osteoimmunology and regulation of inflammatory gene expression. Dysregulated RANK signaling has been associated with abnormal bone remodeling phenotypes and altered immune cell function, making TNFRSF11A a widely used target for mechanistic studies of skeletal and inflammatory biology.

RANK CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous TNFRSF11A expression without altering the underlying DNA sequence.

RANK CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the TNFRSF11A 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 TNFRSF11A transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous RANK expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native TNFRSF11A locus and enabling the study of RANK-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of RANK pathway restoration in tumor cells with silenced or reduced TNFRSF11A expression.

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