CA VIII CRISPR Activation Plasmid (h): sc-403750-ACT

Human CA8 encodes carbonic anhydrase VIII (CA VIII), an acatalytic carbonic anhydrase-related protein that modulates neuronal signaling rather than CO2 hydration. CA VIII is highly enriched in the central nervous system and regulates intracellular calcium dynamics through interaction with inositol 1,4,5-trisphosphate receptor pathways, influencing Purkinje cell excitability and synaptic function. Altered CA8 expression or function has been associated with neurodevelopmental and motor coordination phenotypes, supporting its relevance to cerebellar circuitry and calcium-dependent signaling networks. As a non-enzymatic CA family member, CA VIII also provides a model for dissecting scaffold-like roles of pseudoenzymes in cell signaling.

CA VIII CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous CA8 expression without altering the underlying DNA sequence.

CA VIII CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the CA8 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 CA8 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous CA VIII expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native CA8 locus and enabling the study of CA VIII-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of CA VIII pathway restoration in tumor cells with silenced or reduced CA8 expression.

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