FANCD2 CRISPR Activation Plasmid (h): sc-400445-ACT

FANCD2 encodes a central effector of the Fanconi anemia (FA) DNA interstrand crosslink repair pathway that safeguards genome stability during DNA replication. Following replication stress or crosslink damage, FANCD2 is monoubiquitinated and cooperates with FANCI to coordinate nucleolytic processing, translesion synthesis, and homologous recombination, linking the FA core complex with BRCA-associated repair networks. This activity is tightly coupled to S-phase checkpoint signaling and replication fork protection, helping limit chromosomal breakage and aberrant recombination. Dysregulation or inherited defects in FANCD2 function are associated with Fanconi anemia biology and broader mechanisms of DNA damage sensitivity and tumor suppressor pathway failure in research contexts.

FANCD2 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous FANCD2 expression without altering the underlying DNA sequence.

FANCD2 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the FANCD2 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 FANCD2 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous FANCD2 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native FANCD2 locus and enabling the study of FANCD2-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of FANCD2 pathway restoration in tumor cells with silenced or reduced FANCD2 expression.

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