ALKBH8 CRISPR Activation Plasmid (h): sc-407123-ACT

ALKBH8 encodes a 2-oxoglutarate/Fe(II)-dependent oxygenase implicated in RNA modification, particularly in the maturation of wobble uridine (U34) in specific tRNAs through methylation and subsequent hydroxylation steps. By shaping tRNA decoding efficiency and translational fidelity, ALKBH8 links RNA epitranscriptomic regulation to proteome maintenance and cellular stress responses. Perturbation of ALKBH8 activity can alter codon-biased translation programs and has been associated with dysregulated proliferation and genome stability phenotypes in model systems. These features make ALKBH8 relevant to studies of RNA metabolism, oxidative stress adaptation, and disease-associated translation rewiring.

ALKBH8 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous ALKBH8 expression without altering the underlying DNA sequence.

ALKBH8 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the ALKBH8 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 ALKBH8 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous ALKBH8 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native ALKBH8 locus and enabling the study of ALKBH8-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of ALKBH8 pathway restoration in tumor cells with silenced or reduced ALKBH8 expression.

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