FANCI CRISPR Activation Plasmid (h): sc-402229-ACT

FANCI encodes a key component of the Fanconi anemia (FA) DNA repair pathway that safeguards genome integrity during replication stress. Upon DNA interstrand crosslink damage, FANCI forms the ID complex with FANCD2, undergoes regulatory phosphorylation and monoubiquitination, and coordinates nucleolytic processing with homologous recombination to enable stalled replication fork restart. This signaling axis interfaces with ATR-dependent checkpoint control and replication-coupled repair to limit chromosomal breakage and micronuclei formation. Disruption of FA pathway regulation, including FANCI dysfunction, is associated with inherited genome instability syndromes and is widely studied for its impact on DNA damage sensitivity and mutational burden.

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

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

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