COP1 CRISPR Activation Plasmid (h): sc-402776-ACT

RFWD2 encodes COP1, a RING-type E3 ubiquitin ligase that regulates protein stability by catalyzing ubiquitination and proteasomal turnover of multiple signaling and transcriptional regulators. COP1 influences cellular programs including DNA damage responses, cell-cycle progression, and stress-adaptive transcription by modulating the abundance and activity of pathway effectors. Through its control of ubiquitin-dependent proteostasis, COP1 can reshape transcriptional networks relevant to oncogenic signaling, differentiation, and cellular homeostasis. Dysregulated COP1 expression or activity has been associated with altered tumor suppressor and oncogene balance, making RFWD2 a useful node for mechanistic studies of ubiquitin pathway–linked disease biology.

COP1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous RFWD2 expression without altering the underlying DNA sequence.

COP1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the RFWD2 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 RFWD2 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous COP1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native RFWD2 locus and enabling the study of COP1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of COP1 pathway restoration in tumor cells with silenced or reduced RFWD2 expression.

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