Dhh CRISPR Activation Plasmid (h): sc-403357-ACT

Human DHH encodes Desert hedgehog (Dhh), a secreted morphogen of the Hedgehog family that regulates cell fate specification and tissue patterning through canonical PTCH1–SMO signaling and downstream GLI transcription factors. Dhh is particularly important in peripheral nerve development and gonadal differentiation, coordinating Schwann cell lineage maturation and testicular somatic cell function. Altered DHH expression or pathway activity has been associated with disorders of sex development and peripheral neuropathy-related phenotypes, and it is frequently studied as a context-dependent regulator of developmental signaling programs. As a ligand-driven pathway node, Dhh provides a tractable handle for dissecting paracrine communication, morphogen gradients, and transcriptional responses in stem cell, organoid, and developmental model systems.

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

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

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