KCC1 CRISPR Activation Plasmid (h): sc-405294-ACT

Human SLC12A4 encodes the K+-Cl− cotransporter KCC1, an electroneutral membrane transporter that mediates coupled potassium and chloride efflux to regulate intracellular chloride concentration, cell volume, and osmotic homeostasis. KCC1 activity influences electrochemical gradients that shape membrane potential and chloride-dependent signaling, interfacing with ion transport networks controlled by WNK-SPAK/OSR1 kinase pathways and volume-regulated responses. Through its role in maintaining ionic balance, KCC1 contributes to processes such as proliferation, apoptosis sensitivity, and cellular hydration status across multiple tissues. Dysregulated cation-chloride transport has been implicated in pathological states where volume control and chloride handling are altered, supporting the use of SLC12A4 as a mechanistic node in studies of transporter biology and stress adaptation.

KCC1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous SLC12A4 expression without altering the underlying DNA sequence.

KCC1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the SLC12A4 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 SLC12A4 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous KCC1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native SLC12A4 locus and enabling the study of KCC1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of KCC1 pathway restoration in tumor cells with silenced or reduced SLC12A4 expression.

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