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

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Zhao, D, Jones, JL ORCID: 0000-0002-6475-9134, Gasperini, RJ ORCID: 0000-0001-6859-1247, Charlesworth, JC ORCID: 0000-0001-6201-3518, Liu, G-S ORCID: 0000-0003-3379-724X and Burdon, KP ORCID: 0000-0001-8217-1249 2021 , 'Rapid and efficient cataract gene evaluation in F0 zebrafish using CRISPR-Cas9 ribonucleoprotein complexes' , Methods: A Companion to Methods in Enzymology , doi: 10.1016/j.ymeth.2020.12.004.

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Abstract

Cataract is the leading cause of blindness worldwide. Congenital or paediatric cataract can result in permanentvisual impairment or blindness even with best attempts at treatment. A significant proportion of paediatriccataract has a genetic cause. Therefore, identifying the genes that lead to cataract formation is essential forunderstanding the pathological process of inherited paediatric cataract as well as to the development of newtherapies. 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 evaluatecataract 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 usingbasic laboratory supplies.

Item Type: Article
Authors/Creators:Zhao, D and Jones, JL and Gasperini, RJ and Charlesworth, JC and Liu, G-S and Burdon, KP
Keywords: CRISPR, Cas9, Ribonucleoprotein (RNP) complex, genome editing, cataract, zebrafish
Journal or Publication Title: Methods: A Companion to Methods in Enzymology
Publisher: Academic Press Inc Elsevier Science
ISSN: 1046-2023
DOI / ID Number: 10.1016/j.ymeth.2020.12.004
Copyright Information:

© 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/)

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