AFMID CRISPR Activation Plasmid (h): sc-414235-ACT

Human AFMID (arylformamidase) catalyzes the hydrolysis of N-formylkynurenine to kynurenine in the tryptophan catabolic pathway, supporting flux through the kynurenine–NAD+ biosynthesis axis. By shaping intracellular levels of kynurenine and downstream metabolites, AFMID influences redox homeostasis and metabolic signaling programs coupled to mitochondrial function and cellular stress responses. Dysregulated tryptophan metabolism and altered kynurenine pathway activity have been associated with immune modulation and metabolic remodeling observed across multiple disease contexts, making AFMID a useful node for mechanistic studies of pathway control. AFMID expression and activity are therefore relevant for investigating how nutrient utilization intersects with inflammation-associated signaling and cellular adaptation.

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

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

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