figshare
Browse
143639 - rapid and efficient cataract gene_in press version.pdf (7.86 MB)

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

Download (7.86 MB)
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.

Funding

National Health & Medical Research Council

History

Publication title

Methods: A Companion to Methods in Enzymology

ISSN

1046-2023

Department/School

Menzies Institute for Medical Research

Publisher

Academic Press Inc Elsevier Science

Place of publication

525 B St, Ste 1900, San Diego, USA, Ca, 92101-4495

Rights 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/)

Repository Status

  • Open

Socio-economic Objectives

Diagnosis of human diseases and conditions; Expanding knowledge in the biomedical and clinical sciences

Usage metrics

    University Of Tasmania

    Exports

    RefWorks
    BibTeX
    Ref. manager
    Endnote
    DataCite
    NLM
    DC