RFC1 CRISPR Activation Plasmid (h): sc-402122-ACT

RFC1 encodes replication factor C subunit 1, the large subunit of the RFC clamp loader complex that loads PCNA onto DNA to promote high-processivity DNA polymerase activity. This ATP-dependent process is central to DNA replication, Okazaki fragment maturation, and multiple DNA repair pathways including nucleotide excision repair, base excision repair, and mismatch repair. Through its role in replication stress responses and maintenance of genome stability, RFC1 is frequently studied in contexts of cell-cycle control and DNA damage signaling. Dysregulated RFC1 pathway activity is relevant to proliferative biology and genome instability phenotypes that are commonly modeled in cancer and other DNA repair–associated disorders.

RFC1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous RFC1 expression without altering the underlying DNA sequence.

RFC1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the RFC1 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 RFC1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous RFC1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native RFC1 locus and enabling the study of RFC1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of RFC1 pathway restoration in tumor cells with silenced or reduced RFC1 expression.

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