Intersectin CRISPR Activation Plasmid (h): sc-404804-ACT

Human ITSN1 encodes intersectin, a multidomain adaptor and scaffold protein that integrates endocytic trafficking with signal transduction. Intersectin coordinates clathrin-mediated endocytosis and vesicle dynamics through interactions with dynamin and other endocytic factors, while its DH-PH module links membrane remodeling to Rho-family GTPase signaling and actin cytoskeleton regulation. Through these functions, ITSN1 influences receptor internalization, synaptic vesicle recycling, and downstream pathways including MAPK/ERK and cytoskeletal remodeling. Dysregulated ITSN1 dosage or activity has been associated with neurodevelopmental and neurodegenerative phenotypes and has been studied in the context of altered trafficking and signaling programs in cancer biology.

Intersectin CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous ITSN1 expression without altering the underlying DNA sequence.

Intersectin CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the ITSN1 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 ITSN1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Intersectin expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native ITSN1 locus and enabling the study of Intersectin-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Intersectin pathway restoration in tumor cells with silenced or reduced ITSN1 expression.

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