NOPAR CRISPR Activation Plasmid (h): sc-409016-ACT

MED12L encodes NOPAR, a nuclear protein linked to transcriptional control and RNA polymerase II–dependent gene regulation through Mediator-associated mechanisms. By influencing enhancer–promoter communication and cofactor recruitment, MED12L is positioned to modulate cell-state programs such as proliferation, differentiation, and stress-responsive transcription. Perturbation of Mediator-axis components, including MED12L, is recurrently associated with dysregulated gene-expression networks observed in developmental disorders and diverse cancers, supporting its relevance for mechanistic studies of transcriptional misregulation. MED12L/NOPAR interrogation is therefore useful for mapping regulatory circuitry and connecting genotype-to-phenotype relationships in human cell models.

NOPAR CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous MED12L expression without altering the underlying DNA sequence.

NOPAR CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the MED12L 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 MED12L transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous NOPAR expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native MED12L locus and enabling the study of NOPAR-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of NOPAR pathway restoration in tumor cells with silenced or reduced MED12L expression.

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