HES4 CRISPR Activation Plasmid (h): sc-408938-ACT

Human HES4 encodes a basic helix–loop–helix transcriptional repressor in the Hairy/Enhancer-of-split family that functions as a downstream effector of canonical Notch signaling. By binding E-box–like motifs and recruiting corepressor complexes, HES4 modulates transcriptional programs controlling cell fate decisions, differentiation timing, and maintenance of progenitor-like states. HES4 activity is linked to developmental and lineage-specification processes and intersects with pathways governing stemness and differentiation, making it relevant to studies of dysregulated transcriptional control in cancer and other proliferative disorders. Because Notch–HES axis perturbations can shift differentiation trajectories and alter proliferative capacity, HES4 is frequently investigated as a context-dependent regulator of gene expression networks.

HES4 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous HES4 expression without altering the underlying DNA sequence.

HES4 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the HES4 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 HES4 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous HES4 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native HES4 locus and enabling the study of HES4-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of HES4 pathway restoration in tumor cells with silenced or reduced HES4 expression.

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