NPAS4 CRISPR Activation Plasmid (m): sc-432569-ACT

Mouse Npas4 encodes neuronal PAS domain protein 4 (NPAS4), an activity-dependent basic helix–loop–helix PAS transcription factor that links calcium influx to gene programs controlling inhibitory synapse development and excitatory–inhibitory balance. NPAS4 regulates transcriptional networks involved in synaptic plasticity, circuit refinement, and stimulus-responsive remodeling, integrating signals from neuronal depolarization and downstream CREB-associated pathways. Altered NPAS4 expression has been associated with neuropsychiatric and neurodevelopmental phenotypes in research settings, including stress-related behavioral adaptations, seizure susceptibility, and cognitive dysfunction, highlighting its relevance to mechanisms of network stability. As a rapid early-response regulator, NPAS4 is widely used to probe how neuronal activity reshapes transcriptional states and synaptic connectivity.

NPAS4 CRISPR Activation Plasmid (m) provides a targeted, non-destructive approach to upregulating endogenous Npas4 expression without altering the underlying DNA sequence.

NPAS4 CRISPR Activation Plasmid (m) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the Npas4 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 Npas4 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous NPAS4 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native Npas4 locus and enabling the study of NPAS4-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of NPAS4 pathway restoration in tumor cells with silenced or reduced Npas4 expression.

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