Rab 11A CRISPR Activation Plasmid (h): sc-400617-ACT

RAB11A encodes the small GTPase Rab 11A, a central regulator of recycling endosome dynamics that coordinates vesicle budding, transport, and tethering to the plasma membrane. Rab 11A controls polarized membrane delivery, receptor recycling, cytokinesis, and epithelial lumen formation by engaging Rab11-family interacting proteins (FIPs) and coupling to actin- and microtubule-based motors. Through these trafficking functions, Rab 11A influences signaling outputs from surface receptors and integrins and contributes to endocytic sorting decisions that shape cell migration and polarity. Dysregulated Rab11A-dependent recycling has been linked to phenotypes relevant to oncogenic transformation, neurodegenerative processes, and host–pathogen interactions where membrane trafficking and compartmentalization are perturbed.

Rab 11A CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous RAB11A expression without altering the underlying DNA sequence.

Rab 11A CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the RAB11A 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 RAB11A transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Rab 11A expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native RAB11A locus and enabling the study of Rab 11A-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Rab 11A pathway restoration in tumor cells with silenced or reduced RAB11A expression.

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