CA III CRISPR Activation Plasmid (h): sc-403538-ACT

Human CA3 encodes carbonic anhydrase III (CA III), a cytosolic zinc metalloenzyme that catalyzes the reversible hydration of CO₂ to bicarbonate and protons, contributing to intracellular pH buffering and carbon flux. CA III is highly expressed in skeletal muscle and other oxidative tissues, where it intersects with metabolic homeostasis, redox balance, and responses to oxidative stress through reactive cysteine residues and S-glutathionylation. Altered CA3 expression has been reported in contexts of muscle physiology and metabolic remodeling, supporting its use as a molecular readout in studies of myopathy, energy metabolism, and stress-adaptive programs. As a component of carbonic anhydrase–mediated acid–base regulation, CA III provides a tractable node for probing how pH dynamics influence enzymatic activity, proteostasis, and cellular fitness.

CA III CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous CA3 expression without altering the underlying DNA sequence.

CA III CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the CA3 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 CA3 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous CA III expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native CA3 locus and enabling the study of CA III-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of CA III pathway restoration in tumor cells with silenced or reduced CA3 expression.

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