MGMT CRISPR Activation Plasmid (h): sc-400720-ACT

MGMT (O6-methylguanine-DNA methyltransferase) is a conserved DNA repair protein that removes alkyl adducts from the O6 position of guanine via a direct reversal mechanism, restoring base-pairing fidelity and limiting mutagenesis. As a suicide enzyme that transfers the alkyl group to an active-site cysteine, MGMT influences cellular responses to endogenous and exogenous alkylation stress and interfaces with genome maintenance programs that shape cell cycle progression and DNA damage signaling. Variation in MGMT expression and promoter regulation has been linked to altered susceptibility to mutation accumulation and genomic instability across multiple disease contexts, making it a key node for studying DNA repair capacity. MGMT is also widely used as a functional readout in pathways governing genotoxic stress tolerance and epigenetic control of DNA repair gene expression.

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

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

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