PUBLICATION

A simple and effective F0 knockout method for rapid screening of behaviour and other complex phenotypes

Authors
Kroll, F., Powell, G.T., Ghosh, M., Gestri, G., Antinucci, P., Hearn, T.J., Tunbak, H., Lim, S., Dennis, H.W., Fernandez, J.M., Whitmore, D., Dreosti, E., Wilson, S.W., Hoffman, E.J., Rihel, J.
ID
ZDB-PUB-210109-25
Date
2021
Source
eLIFE   10: (Journal)
Registered Authors
Gestri, Gaia, Hoffman, Ellen, Powell, Gareth, Rihel, Jason, Whitmore, David, Wilson, Steve
Keywords
CRISPR, G0, behaviour, circadian rhythm, genetics, genomics, knockout, neuroscience, sleep, zebrafish
MeSH Terms
  • Animals
  • Behavior, Animal
  • CRISPR-Cas Systems*
  • Embryo, Nonmammalian
  • Gene Knockout Techniques*
  • Genetic Testing/methods*
  • Phenotype
  • RNA, Guide, Kinetoplastida/analysis*
  • Zebrafish/embryology
  • Zebrafish/genetics*
PubMed
33416493 Full text @ Elife
Abstract
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.
Genes / Markers
Figures
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Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Antibodies
Orthology
Engineered Foreign Genes
Mapping