Notch1 CRISPR Activation Plasmid (m): sc-421930-ACT

Mouse Notch1 encodes a single-pass transmembrane receptor that mediates canonical Notch signaling to control cell fate decisions, proliferation, and differentiation across multiple tissues. Ligand engagement triggers proteolytic cleavage and nuclear translocation of the Notch intracellular domain, which partners with RBPJ to regulate transcriptional programs such as Hes and Hey family targets. Notch1 activity intersects with pathways governing stem and progenitor cell maintenance, epithelial-to-mesenchymal dynamics, and immune lineage specification. Dysregulated Notch1 signaling is widely used as a mechanistic model in developmental defects and disease-associated processes including aberrant differentiation and oncogenic transcriptional rewiring.

Notch1 CRISPR Activation Plasmid (m) provides a targeted, non-destructive approach to upregulating endogenous Notch1 expression without altering the underlying DNA sequence.

Notch1 CRISPR Activation Plasmid (m) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the Notch1 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 Notch1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Notch1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native Notch1 locus and enabling the study of Notch1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Notch1 pathway restoration in tumor cells with silenced or reduced Notch1 expression.

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