Back to Search
Start Over
Functional validation of the albinism-associated tyrosinase T373K SNP by CRISPR/Cas9-mediated homology-directed repair (HDR) in rabbits
- Source :
- EBioMedicine
- Publication Year :
- 2018
- Publisher :
- Elsevier, 2018.
-
Abstract
- Background Oculocutaneous albinism (OCA) is a group of autosomal recessive disorders characterized by reduced melanin that are caused by mutations in the gene encoding tyrosinase (TYR), which is the rate-limiting enzyme in the production of the pigment melanin. Many studies or meta-analyses have suggested an association between the TYR T373K SNP and OCA1, but there is limited biochemical and genetic evidence to support this association. Methods We overexpressed TYR-WT and TYR-T373K mutants on HK293T cells and tested the changes of melanin production and tyrosinase activity. Then we generated TYR-K373T knock-in (KI) rabbits by microinjection of ssDNA and synthesized RNAs targeting C1118A using CRISPR/Cas9-HDR to observe the formation of melanin. Findings We demonstrated that the T373K mutation in TYR can reduce tyrosinase activity, leading to an absence of melanin synthesis at the cell-level. The gene-edited TYR-K373T rabbits exhibited rescued melanin production in hair follicles and irises, as inferred from the evident decrease in pigmentation in TYR-T373K rabbits, thus providing functional validation of the albinism-associated T373K SNP at the animal level. Interpretation Our study provides the first animal-level functional validation of the albinism-associated TYR K373T SNP in rabbits, and these results will facilitate gene therapy of OCA1 in pre-clinical settings in the future. Fund The National Key Research and Development Program of China Stem Cell and Translational Research, the Strategic Priority Research Program of the Chinese Academy of Sciences, the Guangdong Province Science and Technology Plan Project, and the Program for JLU Science and Technology Innovative Research Team.
- Subjects :
- 0301 basic medicine
Research paper
Tyrosinase
Mutant
Rabbit
Biology
medicine.disease_cause
Polymorphism, Single Nucleotide
General Biochemistry, Genetics and Molecular Biology
Melanin
Homology directed repair
03 medical and health sciences
medicine
Animals
Genetic Predisposition to Disease
Gene
CRISPR/Cas9
Cells, Cultured
Genetic Association Studies
Genetics
OCA
Gene Editing
Melanins
Mutation
T373K
Point mutation
Monophenol Monooxygenase
Recombinational DNA Repair
General Medicine
medicine.disease
Oculocutaneous albinism
Enzyme Activation
Disease Models, Animal
030104 developmental biology
Amino Acid Substitution
Albinism, Oculocutaneous
Genetic Loci
Albinism
Female
sense organs
Rabbits
CRISPR-Cas Systems
Subjects
Details
- Language :
- English
- ISSN :
- 23523964
- Volume :
- 36
- Database :
- OpenAIRE
- Journal :
- EBioMedicine
- Accession number :
- edsair.doi.dedup.....0674ebe74666aaa2bc6ffff2eba12d74