A simple and effective F0 knockout method for rapid screening of behaviour and other complex phenotypes
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 l...
| Main Authors: | , , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
eLife Sciences Publications Ltd
2021-01-01
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| Series: | eLife |
| Subjects: | |
| Online Access: | https://elifesciences.org/articles/59683 |
| _version_ | 1797367179478302720 |
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| author | François Kroll Gareth T Powell Marcus Ghosh Gaia Gestri Paride Antinucci Timothy J Hearn Hande Tunbak Sumi Lim Harvey W Dennis Joseph M Fernandez David Whitmore Elena Dreosti Stephen W Wilson Ellen J Hoffman Jason Rihel |
| author_facet | François Kroll Gareth T Powell Marcus Ghosh Gaia Gestri Paride Antinucci Timothy J Hearn Hande Tunbak Sumi Lim Harvey W Dennis Joseph M Fernandez David Whitmore Elena Dreosti Stephen W Wilson Ellen J Hoffman Jason Rihel |
| author_sort | François Kroll |
| collection | DOAJ |
| description | 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 knockout crystal fish for imaging. Our F0 knockout method cuts the experimental time from gene to behavioural phenotype in zebrafish from months to one week. |
| first_indexed | 2024-03-08T17:14:36Z |
| format | Article |
| id | doaj.art-25c5de04b6ba4affaa9daf01476b7bbd |
| institution | Directory Open Access Journal |
| issn | 2050-084X |
| language | English |
| last_indexed | 2024-03-08T17:14:36Z |
| publishDate | 2021-01-01 |
| publisher | eLife Sciences Publications Ltd |
| record_format | Article |
| series | eLife |
| spelling | doaj.art-25c5de04b6ba4affaa9daf01476b7bbd2024-01-03T14:41:43ZengeLife Sciences Publications LtdeLife2050-084X2021-01-011010.7554/eLife.59683A simple and effective F0 knockout method for rapid screening of behaviour and other complex phenotypesFrançois Kroll0https://orcid.org/0000-0001-9908-2648Gareth T Powell1Marcus Ghosh2https://orcid.org/0000-0002-2428-4605Gaia Gestri3https://orcid.org/0000-0001-8854-1546Paride Antinucci4https://orcid.org/0000-0003-0573-5383Timothy J Hearn5Hande Tunbak6https://orcid.org/0000-0003-3180-1401Sumi Lim7Harvey W Dennis8Joseph M Fernandez9David Whitmore10Elena Dreosti11https://orcid.org/0000-0002-6738-7057Stephen W Wilson12https://orcid.org/0000-0002-8557-5940Ellen J Hoffman13Jason Rihel14https://orcid.org/0000-0003-4067-2066Department of Cell and Developmental Biology, University College London, London, United KingdomDepartment of Cell and Developmental Biology, University College London, London, United KingdomDepartment of Cell and Developmental Biology, University College London, London, United KingdomDepartment of Cell and Developmental Biology, University College London, London, United KingdomDepartment of Neuroscience, Physiology and Pharmacology, University College London, London, United KingdomDepartment of Cell and Developmental Biology, University College London, London, United KingdomWolfson Institute for Biomedical Research, University College London, London, United KingdomDepartment of Cell and Developmental Biology, University College London, London, United KingdomSchool of Biological Sciences, Faculty of Science, University of Bristol, Bristol, United KingdomChild Study Center, Yale School of Medicine, New Haven, United StatesDepartment of Cell and Developmental Biology, University College London, London, United Kingdom; Department of Molecular and Cell Biology, James Cook University, Townsville, AustraliaWolfson Institute for Biomedical Research, University College London, London, United KingdomDepartment of Cell and Developmental Biology, University College London, London, United KingdomChild Study Center, Yale School of Medicine, New Haven, United States; Department of Neuroscience, Yale School of Medicine, New Haven, United StatesDepartment of Cell and Developmental Biology, University College London, London, United KingdomHundreds 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 knockout crystal fish for imaging. Our F0 knockout method cuts the experimental time from gene to behavioural phenotype in zebrafish from months to one week.https://elifesciences.org/articles/59683behaviourcircadian rhythmsleepknockoutG0CRISPR |
| spellingShingle | François Kroll Gareth T Powell Marcus Ghosh Gaia Gestri Paride Antinucci Timothy J Hearn Hande Tunbak Sumi Lim Harvey W Dennis Joseph M Fernandez David Whitmore Elena Dreosti Stephen W Wilson Ellen J Hoffman Jason Rihel A simple and effective F0 knockout method for rapid screening of behaviour and other complex phenotypes eLife behaviour circadian rhythm sleep knockout G0 CRISPR |
| title | A simple and effective F0 knockout method for rapid screening of behaviour and other complex phenotypes |
| title_full | A simple and effective F0 knockout method for rapid screening of behaviour and other complex phenotypes |
| title_fullStr | A simple and effective F0 knockout method for rapid screening of behaviour and other complex phenotypes |
| title_full_unstemmed | A simple and effective F0 knockout method for rapid screening of behaviour and other complex phenotypes |
| title_short | A simple and effective F0 knockout method for rapid screening of behaviour and other complex phenotypes |
| title_sort | simple and effective f0 knockout method for rapid screening of behaviour and other complex phenotypes |
| topic | behaviour circadian rhythm sleep knockout G0 CRISPR |
| url | https://elifesciences.org/articles/59683 |
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