Fhit CRISPR Activation Plasmid (h): sc-404220-ACT

FHIT encodes Fhit, a histidine triad (HIT) nucleotide-binding protein frequently linked to maintenance of genome integrity and cellular stress responses. Fhit participates in signaling processes that influence DNA damage surveillance, replication stress, and apoptotic pathways, and its activity has been connected to regulation of proliferation and cellular transformation. As a locus within a common fragile site, FHIT is often studied in the context of genomic instability and altered expression patterns observed across diverse tumor-derived models. These features make FHIT a useful target for dissecting pathways that couple nucleotide metabolism and stress signaling to cell-cycle control.

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

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

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