Orexin-A CRISPR Activation Plasmid (h): sc-400357-ACT

Human HCRT encodes prepro-orexin, which is proteolytically processed to generate orexin-A, a neuropeptide that signals primarily through OX1R and OX2R (HCRTR1/HCRTR2) to regulate arousal, sleep–wake stability, feeding behavior, reward processing, and autonomic homeostasis. Orexin-A engages GPCR-driven pathways including Gq/11- and Gi/o-coupled signaling, modulating intracellular calcium dynamics, MAPK/ERK activity, and neuronal excitability across hypothalamic and brainstem circuits. Dysregulation of orexin signaling is strongly linked to sleep disorders such as narcolepsy and broader phenotypes involving metabolic balance and stress responsivity. In biomedical research, HCRT/Orexin-A is used to interrogate neuromodulatory network function, circuit-level integration of energy status, and transcriptional programs associated with arousal and motivation.

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

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

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