adenosine deaminase CRISPR Activation Plasmid (h): sc-402201-ACT

Human ADA encodes adenosine deaminase, a purine salvage enzyme that irreversibly deaminates adenosine and deoxyadenosine to inosine and deoxyinosine, shaping intracellular and extracellular purine pools. By controlling levels of immunomodulatory nucleosides, ADA influences nucleotide metabolism, lymphocyte development, and broader cellular homeostasis linked to DNA replication and stress responses. Dysregulated ADA activity perturbs purine balance and signaling, and ADA is widely studied in contexts where immune cell function and metabolic remodeling intersect. This makes ADA a useful node for interrogating purine metabolism–immune crosstalk and nucleoside-driven regulation of gene expression programs.

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

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

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