RBCK1 CRISPR Activation Plasmid (h): sc-402044-ACT

RBCK1 (also known as HOIL-1) encodes an E3 ubiquitin ligase that functions as an essential component of the LUBAC complex, catalyzing Met1-linked (linear) ubiquitination to regulate NF-κB and MAPK signaling. Through linear ubiquitin chain assembly on key signaling intermediates, RBCK1 helps coordinate innate immune responses, inflammatory signaling, and cell survival decisions downstream of receptors such as TNFR and pattern-recognition receptors. RBCK1 also contributes to protein quality control pathways and ubiquitin-dependent regulation of autophagy-related processes. Disruption or dysregulation of RBCK1 activity has been associated with immunodeficiency and autoinflammatory phenotypes, as well as myopathic and cardiomyopathic features, making it relevant for mechanistic studies of immune homeostasis and stress responses.

RBCK1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous RBCK1 expression without altering the underlying DNA sequence.

RBCK1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the RBCK1 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 RBCK1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous RBCK1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native RBCK1 locus and enabling the study of RBCK1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of RBCK1 pathway restoration in tumor cells with silenced or reduced RBCK1 expression.

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