CA II CRISPR Activation Plasmid (h): sc-401059-ACT

Human CA2 encodes carbonic anhydrase II (CA II), a highly active cytosolic metalloenzyme that catalyzes the reversible hydration of CO₂ to bicarbonate and protons, supporting intracellular pH control and CO₂/bicarbonate transport. By coupling to bicarbonate transporters and ion exchangers, CA II contributes to acid–base homeostasis, epithelial ion movement, and metabolic pH buffering across multiple tissues. Dysregulated carbonic anhydrase activity has been linked to altered cellular acidification and metabolic adaptation, which are frequently studied in contexts such as renal tubular function and tumor microenvironment pH. CA2 is therefore a useful target for investigating CO₂/HCO₃⁻-dependent signaling, ionic homeostasis, and pH-sensitive enzyme networks.

CA II CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous CA2 expression without altering the underlying DNA sequence.

CA II CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the CA2 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 CA2 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous CA II expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native CA2 locus and enabling the study of CA II-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of CA II pathway restoration in tumor cells with silenced or reduced CA2 expression.

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