Tyrosinase CRISPR Activation Plasmid (h): sc-400255-ACT

Human TYR encodes tyrosinase, a copper-dependent oxidase that catalyzes the rate-limiting steps of melanogenesis by converting L-tyrosine to L-DOPA and DOPAquinone, ultimately driving eumelanin and pheomelanin synthesis in melanosomes. TYR activity integrates with melanosome biogenesis and trafficking programs and is coordinated with pigment-cell transcriptional networks, influencing cellular redox balance and UV-responsive stress pathways. Altered TYR function is linked to pigmentation phenotypes and melanin-associated disorders, and it is frequently leveraged as a molecular readout for melanocyte differentiation and melanosome maturation. As a lineage-restricted enzyme, tyrosinase also serves as a useful model for studying organelle-specific enzymology and regulated secretion in pigment cells.

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

Tyrosinase CRISPR Activation Plasmid (h) 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.