BLM hydrolase CRISPR Activation Plasmid (h): sc-404411-ACT

BLMH encodes bleomycin hydrolase, a neutral cysteine protease that participates in intracellular peptide and amino acid turnover and contributes to cellular detoxification of bleomycin-like compounds. The enzyme is implicated in proteostasis-related processes through proteolytic processing of bioactive peptides and can influence redox balance via modulation of homocysteine thiolactone and related metabolites. BLMH activity intersects with pathways affecting protein quality control and stress responses, linking its expression to phenotypes in rapidly proliferating or metabolically stressed cells. Altered BLMH regulation has been associated in the literature with neurodegeneration- and cancer-related contexts, supporting its use as a target for mechanistic studies of protease function and cellular resilience.

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

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

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