AFX1 CRISPR Activation Plasmid (h): sc-402408-ACT

Human FOXO4 encodes the forkhead box O transcription factor AFX1, a stress-responsive regulator that integrates insulin/IGF-1–PI3K–AKT signaling with programs controlling cell-cycle arrest, oxidative stress resistance, metabolism, and apoptosis. AFX1 activity is modulated by phosphorylation-dependent nuclear–cytoplasmic shuttling and cross-talk with MAPK and JNK pathways, shaping transcriptional outputs linked to cellular homeostasis. Through regulation of target genes involved in DNA damage responses and redox balance, FOXO4 contributes to senescence-associated phenotypes and tissue aging processes. Dysregulated FOXO4/AFX1 signaling has been associated with oncogenic pathway remodeling and altered stress tolerance in multiple disease contexts, making it a useful node for mechanistic studies of transcriptional control.

AFX1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous FOXO4 expression without altering the underlying DNA sequence.

AFX1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the FOXO4 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 FOXO4 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous AFX1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native FOXO4 locus and enabling the study of AFX1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of AFX1 pathway restoration in tumor cells with silenced or reduced FOXO4 expression.

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