robo2 CRISPR Activation Plasmid (m): sc-435289-ACT

Mouse Robo2 encodes the roundabout guidance receptor robo2, a transmembrane protein that binds Slit ligands to regulate axon guidance, neuronal migration, and neurite outgrowth during nervous system development. Robo2-mediated signaling influences cytoskeletal dynamics and cell motility through Rho family GTPase pathways, supporting spatial patterning and connectivity in the CNS and PNS. Beyond neurodevelopment, Robo2 is implicated in epithelial morphogenesis and organogenesis, with genetic and expression studies linking altered ROBO2 activity to congenital anomalies and neurodevelopmental phenotypes. These functions make Robo2 a relevant target for dissecting guidance-cue signaling, circuit assembly, and developmental mechanisms in mouse models.

robo2 CRISPR Activation Plasmid (m) provides a targeted, non-destructive approach to upregulating endogenous Robo2 expression without altering the underlying DNA sequence.

robo2 CRISPR Activation Plasmid (m) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the Robo2 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 Robo2 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous robo2 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native Robo2 locus and enabling the study of robo2-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of robo2 pathway restoration in tumor cells with silenced or reduced Robo2 expression.

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