Rubicon CRISPR Activation Plasmid (m): sc-432438-ACT

Mouse 1700021K19Rik encodes Rubicon (RUN domain and cysteine-rich domain containing, Beclin 1-interacting protein), a regulator of membrane trafficking that modulates autophagy and endolysosomal maturation. Rubicon is best known for restraining autophagosome–lysosome fusion through interactions with the Beclin 1–VPS34 class III PI3K complex and for influencing LC3-associated phagocytosis, thereby shaping lysosomal degradation, inflammation, and cellular stress responses. By controlling flux through autophagy and phagocytic pathways, Rubicon can impact mitochondrial quality control, antimicrobial defense, and cytokine production programs relevant to neurodegeneration, metabolic dysfunction, and immune-mediated pathology. These functions make Rubicon a useful node for dissecting how vesicle trafficking and lysosome-dependent turnover govern homeostasis in diverse mouse cell types.

Rubicon CRISPR Activation Plasmid (m) provides a targeted, non-destructive approach to upregulating endogenous 1700021K19Rik expression without altering the underlying DNA sequence.

Rubicon CRISPR Activation Plasmid (m) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the 1700021K19Rik 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 1700021K19Rik transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Rubicon expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native 1700021K19Rik locus and enabling the study of Rubicon-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Rubicon pathway restoration in tumor cells with silenced or reduced 1700021K19Rik expression.

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