AOF1 CRISPR Activation Plasmid (h): sc-405078-ACT

Human KDM1B (AOF1) encodes a FAD-dependent lysine demethylase that preferentially removes H3K4me1/2 marks to shape chromatin accessibility and transcriptional output. By modulating histone methylation states, AOF1 contributes to epigenetic regulation of cell identity programs, including differentiation, genome stability, and germline or early developmental transcriptional control. KDM1B activity interfaces with broader chromatin remodeling and transcriptional repression/activation networks, influencing pathways that govern proliferation and lineage commitment. Dysregulated histone demethylation and altered KDM1B expression patterns have been associated with epigenetic imbalance observed across multiple disease-relevant contexts, including cancer and developmental phenotypes.

AOF1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous KDM1B expression without altering the underlying DNA sequence.

AOF1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the KDM1B 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 KDM1B transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous AOF1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native KDM1B locus and enabling the study of AOF1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of AOF1 pathway restoration in tumor cells with silenced or reduced KDM1B expression.

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