R2 CRISPR Activation Plasmid (h): sc-400624-ACT

Human RRM2 encodes the R2 subunit of ribonucleotide reductase, an essential enzyme that catalyzes the conversion of ribonucleotides to deoxyribonucleotides required for DNA replication and repair. R2 supplies the diferric-tyrosyl radical cofactor that supports catalytic turnover, thereby linking nucleotide metabolism to S phase progression and genome maintenance. RRM2 expression is tightly regulated by cell-cycle and DNA damage response pathways, and its dysregulation is frequently associated with proliferative phenotypes and replication stress. Altered RRM2 activity has been studied in contexts of tumor biology, antiviral responses, and mechanisms that modulate dNTP pool balance and mutagenesis.

R2 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous RRM2 expression without altering the underlying DNA sequence.

R2 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the RRM2 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 RRM2 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous R2 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native RRM2 locus and enabling the study of R2-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of R2 pathway restoration in tumor cells with silenced or reduced RRM2 expression.

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