Dataset Information

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

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.

SUBMITTER: Kroll F

PROVIDER: S-EPMC7793621 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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

Kroll François F Powell Gareth T GT Ghosh Marcus M Gestri Gaia G Antinucci Paride P Hearn Timothy J TJ Tunbak Hande H Lim Sumi S Dennis Harvey W HW Fernandez Joseph M JM Whitmore David D Dreosti Elena E Wilson Stephen W SW Hoffman Ellen J EJ Rihel Jason J

eLife 20210108

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 capa ...[more]

PMID: 33416493

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Project description:BackgroundPhysiologic signals, such as cardiac interbeat intervals, exhibit complex fluctuations. However, capturing important dynamical properties, including nonstationarities may not be feasible from conventional time series graphical representations.MethodsWe introduce a simple-to-implement visualisation method, termed dynamical density delay mapping ("D3-Map" technique) that provides an animated representation of a system's dynamics. The method is based on a generalization of conventional two-dimensional (2D) Poincaré plots, which are scatter plots where each data point, x(n), in a time series is plotted against the adjacent one, x(n?+?1). First, we divide the original time series, x(n) (n?=?1,…, N), into a sequence of segments (windows). Next, for each segment, a three-dimensional (3D) Poincaré surface plot of x(n), x(n?+?1), h[x(n),x(n?+?1)] is generated, in which the third dimension, h, represents the relative frequency of occurrence of each (x(n),x(n?+?1)) point. This 3D Poincaré surface is then chromatised by mapping the relative frequency h values onto a colour scheme. We also generate a colourised 2D contour plot from each time series segment using the same colourmap scheme as for the 3D Poincaré surface. Finally, the original time series graph, the colourised 3D Poincaré surface plot, and its projection as a colourised 2D contour map for each segment, are animated to create the full "D3-Map."ResultsWe first exemplify the D3-Map method using the cardiac interbeat interval time series from a healthy subject during sleeping hours. The animations uncover complex dynamical changes, such as transitions between states, and the relative amount of time the system spends in each state. We also illustrate the utility of the method in detecting hidden temporal patterns in the heart rate dynamics of a patient with atrial fibrillation. The videos, as well as the source code, are made publicly available.ConclusionsAnimations based on density delay maps provide a new way of visualising dynamical properties of complex systems not apparent in time series graphs or standard Poincaré plot representations. Trainees in a variety of fields may find the animations useful as illustrations of fundamental but challenging concepts, such as nonstationarity and multistability. For investigators, the method may facilitate data exploration.

Dataset Information

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

Publications

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

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