NNMT CRISPR Activation Plasmid (h): sc-403192-ACT

Human NNMT (nicotinamide N-methyltransferase) is a cytosolic methyltransferase that catalyzes N-methylation of nicotinamide and related pyridines using S-adenosyl-L-methionine, linking xenobiotic metabolism to methyl donor homeostasis. By influencing SAM/SAH balance and nicotinamide/NAD⁺-related metabolic flux, NNMT can modulate epigenetic methylation capacity and broader metabolic state. Altered NNMT expression has been associated with metabolic reprogramming and changes in cellular differentiation and stress responses across multiple disease contexts, making it a useful node for studying metabolism–epigenome coupling. NNMT is therefore frequently investigated in pathways related to one-carbon metabolism, redox regulation, and transcriptional control.

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

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

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