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Rapid and efficient cataract gene evaluation in F0 zebrafish using CRISPR-Cas9 ribonucleoprotein complexes

Citation

Zhao, D and Jones, JL and Gasperini, RJ and Charlesworth, JC and Liu, G-S and Burdon, KP, Rapid and efficient cataract gene evaluation in F0 zebrafish using CRISPR-Cas9 ribonucleoprotein complexes, Methods: A Companion to Methods in Enzymology ISSN 1046-2023 (2021) [Refereed Article]


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Copyright Statement

2021 The Authors. Published by Elsevier Inc. This is an open access article under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0)license (https://creativecommons.org/licenses/by-nc-nd/4.0/)

DOI: doi:10.1016/j.ymeth.2020.12.004

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.

Item Details

Item Type:Refereed Article
Keywords:CRISPR, Cas9, Ribonucleoprotein (RNP) complex, genome editing, cataract, zebrafish
Research Division:Biological Sciences
Research Group:Genetics
Research Field:Genetics not elsewhere classified
Objective Division:Expanding Knowledge
Objective Group:Expanding knowledge
Objective Field:Expanding knowledge in the biomedical and clinical sciences
UTAS Author:Zhao, D (Ms Duran Zhao)
UTAS Author:Jones, JL (Dr Johanna Jones)
UTAS Author:Gasperini, RJ (Dr Rob Gasperini)
UTAS Author:Charlesworth, JC (Dr Jac Charlesworth)
UTAS Author:Liu, G-S (Associate Professor Guei-Sheung Liu)
UTAS Author:Burdon, KP (Professor Kathryn Burdon)
ID Code:143639
Year Published:2021
Funding Support:National Health and Medical Research Council (1185477)
Web of Science® Times Cited:2
Deposited By:Menzies Institute for Medical Research
Deposited On:2021-03-29
Last Modified:2021-05-04
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