Heat-shock-induced tyrosinase gene ablation with CRISPR in zebrafish

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 t...

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Bibliographic Details
Published in:Molecular genetics and genomics : MGG 2020-07, Vol.295 (4), p.911-922
Main Authors: Wu, Yu-Ching, Wang, I-Jong
Format: Article
Language:English
Subjects:
Animal Genetics and Genomics
Biochemistry
Biomedical and Life Sciences
CRISPR
Danio rerio
Dihydroxyphenylalanine
Embryos
Eye diseases
Green fluorescent protein
Heat shock
Human Genetics
Levodopa
Life Sciences
Melanin
Microbial Genetics and Genomics
Original Article
Phenotypes
Plant Genetics and Genomics
Ribonucleic acid
RNA
Skin diseases
Tyrosinase gene
Tyrosine
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Summary: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.
ISSN:1617-4615
1617-4623
DOI:10.1007/s00438-020-01681-x