CD73 CRISPR Activation Plasmid (h): sc-400307-ACT

NT5E encodes CD73 (ecto-5′-nucleotidase), a GPI-anchored ectoenzyme that hydrolyzes extracellular AMP to adenosine and inorganic phosphate, shaping purinergic signaling in the tissue microenvironment. By controlling local adenosine availability, CD73 modulates cAMP-linked GPCR pathways and influences immune cell activation, vascular permeability, and platelet–endothelial interactions. CD73 activity contributes to regulation of inflammation and hypoxia-associated responses and is widely used as a marker of stromal and endothelial cell states. Dysregulated NT5E/CD73 expression or function has been associated with altered immune regulation and aberrant tissue remodeling in multiple disease-relevant contexts.

CD73 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous NT5E expression without altering the underlying DNA sequence.

CD73 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the NT5E 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 NT5E transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous CD73 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native NT5E locus and enabling the study of CD73-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of CD73 pathway restoration in tumor cells with silenced or reduced NT5E expression.

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