RFC5 CRISPR Activation Plasmid (h): sc-405098-ACT

Human RFC5 encodes the 36 kDa subunit of replication factor C, an ATP-dependent clamp loader complex that deposits PCNA onto primed DNA to support processive DNA synthesis. RFC5 is required for coordinated DNA replication and repair, contributing to S-phase progression, replication fork stability, and responses to DNA damage through pathways linked to PCNA-dependent polymerase activity and post-replication repair. Because clamp loading and PCNA cycling influence genome integrity, perturbation of RFC5 expression is commonly examined in the context of replication stress, checkpoint signaling, and chromosomal instability phenotypes relevant to cancer biology and other proliferative disorders. RFC5 is therefore a useful node for dissecting mechanisms that couple DNA synthesis with repair and cell-cycle control.

RFC5 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous RFC5 expression without altering the underlying DNA sequence.

RFC5 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the RFC5 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 RFC5 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous RFC5 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native RFC5 locus and enabling the study of RFC5-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of RFC5 pathway restoration in tumor cells with silenced or reduced RFC5 expression.

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