Intersectin-2 CRISPR Activation Plasmid (h): sc-402531-ACT

ITSN2 encodes intersectin-2, a multi-domain endocytic scaffold that integrates clathrin-mediated membrane trafficking with signaling outputs through SH3-mediated interactions with regulators of dynamin, actin remodeling, and small GTPases. By coordinating endocytosis, vesicle recycling, and cytoskeletal dynamics, intersectin-2 contributes to receptor internalization, synaptic and neuronal trafficking programs, and spatial control of signal transduction. ITSN2-associated network perturbations have been linked in the literature to altered growth factor signaling and defects in membrane dynamics that are relevant to cancer biology and neurodevelopmental processes. Its modular architecture makes ITSN2 a useful node for dissecting crosstalk between trafficking pathways and kinase-driven signaling cascades.

Intersectin-2 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous ITSN2 expression without altering the underlying DNA sequence.

Intersectin-2 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the ITSN2 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 ITSN2 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Intersectin-2 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native ITSN2 locus and enabling the study of Intersectin-2-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Intersectin-2 pathway restoration in tumor cells with silenced or reduced ITSN2 expression.

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