A simple and effective F0 knockout method for rapid screening of behaviour and other complex phenotypes
| Autor: | Timothy J Hearn, Stephen W. Wilson, Elena Dreosti, Joseph M Fernandez, Sumi Lim, Marcus Ghosh, Jason Rihel, Gaia Gestri, Paride Antinucci, David Whitmore, Ellen J. Hoffman, François Kroll, Gareth T. Powell, Harvey W Dennis, Hande Tunbak |
|---|---|
| Přispěvatelé: | Kroll, François [0000-0001-9908-2648], Ghosh, Marcus [0000-0002-2428-4605], Gestri, Gaia [0000-0001-8854-1546], Antinucci, Paride [0000-0003-0573-5383], Tunbak, Hande [0000-0003-3180-1401], Dreosti, Elena [0000-0002-6738-7057], Wilson, Stephen W [0000-0002-8557-5940], Rihel, Jason [0000-0003-4067-2066], Apollo - University of Cambridge Repository |
| Rok vydání: | 2021 |
| Předmět: |
circadian rhythm
0301 basic medicine Embryo Nonmammalian QH301-705.5 Science Circadian clock knockout Genomics Computational biology 0601 Biochemistry and Cell Biology General Biochemistry Genetics and Molecular Biology neuroscience Gene Knockout Techniques 03 medical and health sciences 0302 clinical medicine genomics G0 Animals CRISPR genetics Genetic Testing sleep Biology (General) Zebrafish Gene Gene knockout Behavior Animal General Immunology and Microbiology biology General Neuroscience Genetics and Genomics General Medicine biology.organism_classification Phenotype behaviour 3. Good health 030104 developmental biology Medicine CRISPR-Cas Systems 030217 neurology & neurosurgery Research Article RNA Guide Kinetoplastida Genetic screen |
| Zdroj: | eLife, Vol 10 (2021) eLife |
| ISSN: | 2050-084X |
| Popis: | Hundreds of human genes are associated with neurological diseases, but translation into tractable biological mechanisms is lagging. Larval zebrafish are an attractive model to investigate genetic contributions to neurological diseases. However, current CRISPR-Cas9 methods are difficult to apply to large genetic screens studying behavioural phenotypes. To facilitate rapid genetic screening, we developed a simple sequencing-free tool to validate gRNAs and a highly effective CRISPR-Cas9 method capable of converting >90% of injected embryos directly into F0 biallelic knockouts. We demonstrate that F0 knockouts reliably recapitulate complex mutant phenotypes, such as altered molecular rhythms of the circadian clock, escape responses to irritants, and multi-parameter day-night locomotor behaviours. The technique is sufficiently robust to knockout multiple genes in the same animal, for example to create the transparent triple knockoutcrystalfish for imaging. Our F0 knockout method cuts the experimental time from gene to behavioural phenotype in zebrafish from months to one week. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|