SmcY CRISPR Activation Plasmid (h): sc-403217-ACT

KDM5D encodes the Y chromosome–linked lysine demethylase SmcY (JARID1D), a chromatin regulator that removes H3K4me2/3 marks to modulate transcriptional programs. Through its role in epigenetic control, SmcY contributes to maintenance of cell identity, regulation of proliferation-associated gene expression, and coordination of chromatin state with DNA-templated processes such as replication and repair. Altered KDM5 family activity has been implicated in dysregulated transcriptional networks across cancer and developmental contexts, and KDM5D itself is frequently studied in relation to sex-specific biology and Y chromosome loss in somatic tissues. These features make KDM5D a useful model for probing how histone demethylation shapes pathway outputs and cellular phenotypes.

SmcY CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous KDM5D expression without altering the underlying DNA sequence.

SmcY CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the KDM5D 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 KDM5D transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous SmcY expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native KDM5D locus and enabling the study of SmcY-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of SmcY pathway restoration in tumor cells with silenced or reduced KDM5D expression.

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