DAAO CRISPR Activation Plasmid (h): sc-402772-ACT

Human DAO encodes D-amino acid oxidase (DAAO), a peroxisomal flavoprotein that catalyzes oxidative deamination of neutral D-amino acids, generating corresponding imino acids, hydrogen peroxide, and ammonia. By regulating levels of D-serine and related metabolites, DAAO influences NMDA receptor co-agonist availability and links amino acid metabolism to redox balance through peroxisome-associated reactive oxygen species handling. This axis intersects with neurotransmission, cellular oxidative stress responses, and metabolic homeostasis in brain and peripheral tissues. Altered DAAO activity or expression has been investigated in the context of neuropsychiatric and neurodegenerative disease biology, as well as broader models of oxidative damage and metabolic dysregulation.

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

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

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