CA IV CRISPR Activation Plasmid (h): sc-404215-ACT

Human CA4 encodes carbonic anhydrase IV (CA IV), a glycosylphosphatidylinositol-anchored, extracellular metalloenzyme that catalyzes the reversible hydration of CO₂ to bicarbonate and protons. By accelerating CO₂/HCO₃⁻ interconversion at the cell surface, CA IV supports acid–base homeostasis, epithelial ion transport, and CO₂ handling in tissues such as kidney and lung, and contributes to pH regulation in microenvironments shaped by metabolism and perfusion. CA4 activity interfaces with bicarbonate transport systems and pH-sensitive signaling processes that influence cellular physiology, including modulation of transporter function and extracellular buffering capacity. Dysregulated carbonic anhydrase activity and altered pH control are relevant to disease-associated phenotypes in renal and pulmonary physiology and to contexts where extracellular acidification impacts cell behavior.

CA IV CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous CA4 expression without altering the underlying DNA sequence.

CA IV CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the CA4 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 CA4 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous CA IV expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native CA4 locus and enabling the study of CA IV-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of CA IV pathway restoration in tumor cells with silenced or reduced CA4 expression.

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