Tyrosinase CRISPR Activation Plasmid (m): sc-423566-ACT

Mouse Tyr encodes tyrosinase, a copper-dependent oxidoreductase that catalyzes key rate-limiting steps in eumelanin and pheomelanin biosynthesis, including the conversion of L-tyrosine to DOPA and DOPAquinone. Tyrosinase activity underlies melanocyte differentiation programs and links melanogenesis to redox balance, melanosome biogenesis, and pigment granule maturation. Altered Tyr expression or enzymatic function is closely associated with pigmentation phenotypes and is frequently used as a molecular readout in studies of melanocyte biology and melanin pathway regulation. As a lineage-restricted enzyme with measurable biochemical outputs, tyrosinase provides a tractable node for interrogating pigment-associated signaling networks and stress responses.

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

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

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