ESET CRISPR Activation Plasmid (h): sc-401325-ACT

Human SETDB1 encodes ESET, a SET domain–containing histone lysine methyltransferase that deposits repressive H3K9me3 marks to promote heterochromatin formation and stable gene silencing. ESET functions in epigenetic regulation of transcription, suppression of endogenous retroelements, and maintenance of genome integrity through interactions with corepressors and chromatin remodeling complexes. By shaping lineage-specific transcriptional programs, SETDB1 influences pluripotency, differentiation, and cell cycle–associated chromatin states. Dysregulated SETDB1 activity and altered H3K9 methylation landscapes are frequently studied in the context of oncogenic transcriptional reprogramming and immune evasion phenotypes, as well as broader epigenome instability in human disease models.

ESET CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous SETDB1 expression without altering the underlying DNA sequence.

ESET CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the SETDB1 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 SETDB1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous ESET expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native SETDB1 locus and enabling the study of ESET-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of ESET pathway restoration in tumor cells with silenced or reduced SETDB1 expression.

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