CA IX CRISPR Activation Plasmid (h): sc-400905-ACT

Human CA9 encodes carbonic anhydrase IX (CA IX), a hypoxia-inducible, membrane-associated enzyme that catalyzes reversible CO₂ hydration to regulate extracellular acidification and intracellular pH homeostasis. CA IX supports cellular adaptation to low-oxygen stress and interfaces with HIF-1 signaling, ion transport networks, and metabolic reprogramming that influence cell survival, migration, and microenvironmental buffering. Dysregulated CA9 expression is frequently studied as a marker of hypoxic, acidified niches and is linked to altered redox balance and invasion-associated phenotypes in solid-tissue disease models. These properties make CA IX a useful node for investigating pH-dependent signaling, hypoxia-responsive transcriptional programs, and microenvironment-driven selection pressures.

CA IX CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous CA9 expression without altering the underlying DNA sequence.

CA IX CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the CA9 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 CA9 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous CA IX expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native CA9 locus and enabling the study of CA IX-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of CA IX pathway restoration in tumor cells with silenced or reduced CA9 expression.

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