Mi2-b CRISPR Activation Plasmid (h): sc-402185-ACT

CHD4 (Mi2-b) encodes an ATP-dependent helicase that serves as a catalytic core subunit of the NuRD chromatin remodeling and histone deacetylase complex. By coupling nucleosome repositioning with transcriptional repression, Mi2-b helps regulate lineage-specific gene expression programs, DNA replication timing, and chromatin accessibility. CHD4 participates in genome maintenance processes including DNA damage signaling and repair pathway choice, influencing cell-cycle progression and replication stress responses. Dysregulated CHD4/NuRD activity and CHD4 alterations have been associated with aberrant epigenetic states in cancer biology and with neurodevelopmental and congenital disorder phenotypes, supporting its relevance for mechanistic studies of chromatin-driven gene regulation.

Mi2-b CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous CHD4 expression without altering the underlying DNA sequence.

Mi2-b CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the CHD4 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 CHD4 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Mi2-b expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native CHD4 locus and enabling the study of Mi2-b-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Mi2-b pathway restoration in tumor cells with silenced or reduced CHD4 expression.

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