Histone H2A.X CRISPR Activation Plasmid (h): sc-400538-ACT

H2AFX encodes the histone variant Histone H2A.X, a core component of chromatin that becomes rapidly phosphorylated at Ser139 (γH2AX) in response to DNA double-strand breaks. This modification nucleates DNA damage response signaling by recruiting mediator and repair factors, coordinating ATM/ATR kinase pathways, chromatin remodeling, checkpoint activation, and repair through non-homologous end joining and homologous recombination. H2A.X dynamics influence replication stress tolerance, telomere stability, and genome-wide transcriptional programs linked to chromatin integrity. Dysregulation of H2AFX-associated signaling is frequently studied in contexts of genomic instability, oncogenic stress, and sensitivity to genotoxic exposures.

Histone H2A.X CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous H2AFX expression without altering the underlying DNA sequence.

Histone H2A.X CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the H2AFX 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 H2AFX transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Histone H2A.X expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native H2AFX locus and enabling the study of Histone H2A.X-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Histone H2A.X pathway restoration in tumor cells with silenced or reduced H2AFX expression.

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