PUBLICATION
Rapid and efficient cataract gene evaluation in F0 zebrafish using CRISPR-Cas9 ribonucleoprotein complexes
- Authors
- Zhao, D., Jones, J.L., Gasperini, R.J., Charlesworth, J.C., Liu, G.S., Burdon, K.P.
- ID
- ZDB-PUB-210109-30
- Date
- 2021
- Source
- Methods (San Diego, Calif.) 194: 37-47 (Journal)
- Registered Authors
- Keywords
- CRISPR, Cas9, Cataract, Genome editing, Ribonucleoprotein (RNP) complex, Zebrafish
- MeSH Terms
-
- Animals
- Blindness
- CRISPR-Cas Systems
- Cataract*/genetics
- Gene Editing
- Humans
- Ribonucleoproteins/genetics
- Ribonucleoproteins/metabolism
- Zebrafish*/genetics
- Zebrafish*/metabolism
- PubMed
- 33418061 Full text @ Methods
Citation
Zhao, D., Jones, J.L., Gasperini, R.J., Charlesworth, J.C., Liu, G.S., Burdon, K.P. (2021) Rapid and efficient cataract gene evaluation in F0 zebrafish using CRISPR-Cas9 ribonucleoprotein complexes. Methods (San Diego, Calif.). 194:37-47.
Abstract
Cataract is the leading cause of blindness worldwide. Congenital or paediatric cataract can result in permanent visual impairment or blindness even with best attempts at treatment. A significant proportion of paediatric cataract has a genetic cause. Therefore, identifying the genes that lead to cataract formation is essential for understanding the pathological process of inherited paediatric cataract as well as to the development of new therapies. Despite clear progress in genomics technologies, verification of the biological effects of newly identified candidate genes and variants is still challenging. Here, we provide a step-by-step pipeline to evaluate cataract candidate genes in F0 zebrafish using CRISPR-Cas9 ribonucleoprotein complexes (RNP). Detailed descriptions of CRISPR-Cas9 RNP design and formulation, microinjection, optimization of CRISPR-Cas9 RNP reagent dose and delivery route, editing efficacy analysis as well as cataract formation evaluation are included. Following this protocol, any cataract candidates can be readily and efficiently evaluated within 2 weeks using basic laboratory supplies.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping