HCN4 CRISPR Activation Plasmid (h): sc-402293-ACT

HCN4 encodes the hyperpolarization-activated cyclic nucleotide-gated channel 4, a principal mediator of the “funny” current (If) that contributes to spontaneous diastolic depolarization and rhythmic electrical activity. The channel conducts mixed Na⁺/K⁺ inward currents activated by hyperpolarization and is directly modulated by cAMP, linking autonomic signaling to membrane excitability. HCN4 functions at the intersection of pacemaker regulation, membrane repolarization dynamics, and cAMP-dependent signaling pathways that shape action potential firing. Altered HCN4 expression or function has been associated with cardiac conduction phenotypes and rhythm regulation, making it relevant for mechanistic studies of electrophysiology and excitability in human cellular models.

HCN4 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous HCN4 expression without altering the underlying DNA sequence.

HCN4 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the HCN4 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 HCN4 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous HCN4 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native HCN4 locus and enabling the study of HCN4-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of HCN4 pathway restoration in tumor cells with silenced or reduced HCN4 expression.

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