DDAH II CRISPR Activation Plasmid (h): sc-403032-ACT

Human DDAH2 encodes dimethylarginine dimethylaminohydrolase II (DDAH II), a cytosolic enzyme that hydrolyzes asymmetric dimethylarginine (ADMA) and L-NMMA to citrulline and dimethylamine, thereby regulating nitric oxide synthase activity and nitric oxide (NO) bioavailability. Through control of methylarginine metabolism, DDAH II integrates into endothelial signaling, redox-sensitive processes, and vascular homeostasis pathways that influence smooth muscle tone and inflammatory signaling. Altered DDAH2 expression or activity has been linked to impaired NO signaling and endothelial dysfunction phenotypes. These mechanisms make DDAH2 a relevant target for studying cardiometabolic stress responses, vascular inflammation, and NO-dependent cellular programs in human model systems.

DDAH II CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous DDAH2 expression without altering the underlying DNA sequence.

DDAH II CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the DDAH2 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 DDAH2 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous DDAH II expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native DDAH2 locus and enabling the study of DDAH II-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of DDAH II pathway restoration in tumor cells with silenced or reduced DDAH2 expression.

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