FKHR/FOXO1 CRISPR Activation Plasmid (h): sc-400137-ACT

FOXO1 (FKHR) encodes a forkhead box transcription factor that integrates insulin/IGF-1 and growth factor signals to coordinate cell-cycle control, apoptosis, oxidative stress responses, and metabolic homeostasis. FOXO1 activity is regulated by post-translational modifications and subcellular localization downstream of PI3K–AKT signaling, with additional inputs from AMPK and stress-activated pathways. In the nucleus, FOXO1 regulates transcriptional programs involved in gluconeogenesis, autophagy, and differentiation, shaping context-dependent cellular fate decisions. Dysregulated FOXO1 signaling is implicated in insulin resistance and metabolic disease, altered immune cell function, and oncogenic processes including the PAX3–FOXO1 fusion observed in alveolar rhabdomyosarcoma.

FKHR/FOXO1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous FOXO1 expression without altering the underlying DNA sequence.

FKHR/FOXO1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the FOXO1 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 FOXO1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous FKHR/FOXO1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native FOXO1 locus and enabling the study of FKHR/FOXO1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of FKHR/FOXO1 pathway restoration in tumor cells with silenced or reduced FOXO1 expression.

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