mAChR M4 CRISPR Activation Plasmid (h): sc-402318-ACT

CHRM4 encodes the human muscarinic acetylcholine receptor M4 (mAChR M4), a Gi/o-coupled GPCR that modulates neuronal excitability and synaptic transmission by inhibiting adenylyl cyclase, reducing cAMP signaling, and regulating ion channel activity. M4 participates in cholinergic neurotransmission pathways and shapes dopaminergic circuit output through presynaptic and postsynaptic signaling mechanisms. Downstream effects include modulation of MAPK/ERK signaling, calcium dynamics, and neurotransmitter release, linking CHRM4 activity to network-level control of motor and cognitive processes. Altered muscarinic receptor signaling and CHRM4-associated circuit dysregulation have been studied in neuropsychiatric and movement disorder research, including contexts relevant to schizophrenia, Parkinsonian circuitry, and substance use–related phenotypes.

mAChR M4 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous CHRM4 expression without altering the underlying DNA sequence.

mAChR M4 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the CHRM4 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 CHRM4 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous mAChR M4 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native CHRM4 locus and enabling the study of mAChR M4-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of mAChR M4 pathway restoration in tumor cells with silenced or reduced CHRM4 expression.

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