Dnmt2 CRISPR Activation Plasmid (h): sc-402709-ACT

TRDMT1 encodes Dnmt2, an evolutionarily conserved cytosine-5 methyltransferase that primarily methylates specific tRNAs, most notably tRNAAsp, to support RNA stability and proteostasis under cellular stress. Through RNA methylation, Dnmt2 contributes to translational fidelity, stress-response signaling, and maintenance of epitranscriptomic regulation that can influence cell growth and differentiation programs. Altered TRDMT1 activity has been linked to changes in RNA modification patterns associated with genome stability, apoptosis, and susceptibility to stress-induced damage. Dysregulation of these processes is relevant to mechanistic studies of cancer biology, neurobiology, and inflammatory signaling where RNA modification and translation control are frequently perturbed.

Dnmt2 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous TRDMT1 expression without altering the underlying DNA sequence.

Dnmt2 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the TRDMT1 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 TRDMT1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Dnmt2 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native TRDMT1 locus and enabling the study of Dnmt2-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Dnmt2 pathway restoration in tumor cells with silenced or reduced TRDMT1 expression.

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