ALKBH3 CRISPR Activation Plasmid (h): sc-406896-ACT

ALKBH3 (also known as ABH3) encodes a Fe(II)/2-oxoglutarate–dependent dioxygenase that demethylates alkylated nucleic acids, including lesions such as 1-methyladenine and 3-methylcytosine in single-stranded DNA and RNA. By reversing cytotoxic alkylation damage, ALKBH3 contributes to genome maintenance, replication stress tolerance, and RNA metabolism under genotoxic conditions. Its activity interfaces with DNA damage response pathways and can influence transcriptional programs linked to cell-cycle progression. Altered ALKBH3 expression has been reported in multiple tumor contexts and is studied for its relationship to genomic instability phenotypes and cellular sensitivity to alkylating stress in model systems.

ALKBH3 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous ALKBH3 expression without altering the underlying DNA sequence.

ALKBH3 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the ALKBH3 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 ALKBH3 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous ALKBH3 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native ALKBH3 locus and enabling the study of ALKBH3-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of ALKBH3 pathway restoration in tumor cells with silenced or reduced ALKBH3 expression.

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