PUBLICATION
Heat-shock-induced tyrosinase gene ablation with CRISPR in zebrafish
- Authors
- Wu, Y.C., Wang, I.J.
- ID
- ZDB-PUB-200506-8
- Date
- 2020
- Source
- Molecular genetics and genomics : MGG 295(4): 911-922 (Journal)
- Registered Authors
- Wang, I-Jong, Wu, Yu-Ching
- Keywords
- CRISPR, Heat-shock-induced tyrosinase gene ablation, Zebrafish
- MeSH Terms
-
- Animals
- Animals, Genetically Modified
- CRISPR-Associated Protein 9/genetics
- CRISPR-Cas Systems/genetics*
- Embryo, Nonmammalian
- Embryonic Development
- Gene Expression Regulation, Enzymologic
- Gene Knockout Techniques
- Germ-Line Mutation
- Green Fluorescent Proteins/genetics
- Heat-Shock Response/genetics*
- Melanins/biosynthesis
- Melanins/genetics
- Monophenol Monooxygenase/genetics*
- Myosin Light Chains/genetics
- Promoter Regions, Genetic/genetics
- RNA/genetics
- Zebrafish/genetics*
- Zebrafish/growth & development
- Zebrafish Proteins/genetics
- PubMed
- 32367255 Full text @ Mol. Genet. Genomics
Citation
Wu, Y.C., Wang, I.J. (2020) Heat-shock-induced tyrosinase gene ablation with CRISPR in zebrafish. Molecular genetics and genomics : MGG. 295(4):911-922.
Abstract
Tyrosinase (TYR) converts L-tyrosine into 3,4-dihydroxyphenylalanine (L-DOPA) and L-DOPA into L-dopaquinone, which can produce melanin pigment. The abrogation of the functional activity of TYR can result in albino skin and eye diseases because of a deficiency in melanin pigment production. In this study, we developed and characterized an inducible knockout TYR platform comprising the heat-inducible heat-shock-promoter-70-driving CRISPR/Cas9 system and a zU6-promoter-driving tyr single guide RNA (sgRNA) system to investigate the temporal expression of TYR genes. To overcome the difficulty of identifying zebrafish germline integrations and facilitate the observation of Cas9 expression, heart-specific cmlc2:enhanced green fluorescent protein (EGFP; used to confirm tyr sgRNA expression) and two selectable markers (P2A-mCherry and internal ribosomal entry site-EGFP) were applied in our system. Heat shock treatment administered to Cas9 transgenic embryos induced mCherry or EGFP fluorescence expression throughout the embryos' bodies, and Cas9 protein was detected 1 h after heat shock treatment. Mutations were created by direct injection and line crossing, which led to mosaic and complete depigmentation phenotypes in approximately 50% and 100% of the embryos, respectively. Using our system, conditional TYR knockout in zebrafish was achieved efficiently and simply.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping