DOT1L1 CRISPR Activation Plasmid (h): sc-403286-ACT

Human DOT1L (DOT1L1) encodes a histone lysine methyltransferase that catalyzes H3K79 mono-, di-, and trimethylation, an epigenetic mark linked to active transcription within gene bodies. DOT1L-dependent chromatin regulation supports RNA polymerase II elongation, enhancer–promoter communication, and coordination of DNA damage signaling with cell-cycle progression. Through control of lineage-specific transcriptional programs, DOT1L influences hematopoietic differentiation and broader developmental gene networks. Dysregulated DOT1L activity and altered H3K79 methylation states are associated with transcriptional miswiring observed in leukemia and other cancers, as well as context-dependent effects on genome stability.

DOT1L1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous DOT1L expression without altering the underlying DNA sequence.

DOT1L1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the DOT1L 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 DOT1L transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous DOT1L1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native DOT1L locus and enabling the study of DOT1L1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of DOT1L1 pathway restoration in tumor cells with silenced or reduced DOT1L expression.

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