KCC2 CRISPR Activation Plasmid (h): sc-402080-ACT

Human SLC12A5 encodes the neuron-specific K+/Cl− cotransporter KCC2, a key regulator of intracellular chloride homeostasis that shapes the polarity and efficacy of GABAergic and glycinergic neurotransmission. By coupling K+ and Cl− extrusion, KCC2 supports inhibitory synaptic signaling, neuronal maturation, and activity-dependent plasticity, and it interfaces with cytoskeletal and trafficking processes that control membrane localization. Altered KCC2 expression or function has been linked to disrupted excitation–inhibition balance and network hyperexcitability in multiple neurodevelopmental and neurological disease contexts. As a result, SLC12A5 is widely studied in pathways governing synaptic inhibition, neuronal differentiation, and circuit stability.

KCC2 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous SLC12A5 expression without altering the underlying DNA sequence.

KCC2 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the SLC12A5 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 SLC12A5 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous KCC2 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native SLC12A5 locus and enabling the study of KCC2-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of KCC2 pathway restoration in tumor cells with silenced or reduced SLC12A5 expression.

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