A simple and effective F0 knockout method for rapid screening of behaviour and other complex phenotypes.
A simple and effective F0 knockout method for rapid screening of behaviour and other complex phenotypes.
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DOI：
10.7554/elife.59683
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发表时间：
2021-01-08
期刊：
影响因子：
7.7
通讯作者：
Rihel J
中科院分区：
文献类型：
--
作者：
Kroll F;Powell GT;Ghosh M;Gestri G;Antinucci P;Hearn TJ;Tunbak H;Lim S;Dennis HW;Fernandez JM;Whitmore D;Dreosti E;Wilson SW;Hoffman EJ;Rihel J
数百个人类基因与神经系统疾病有关，但转化为易处理的生物机制却很滞后。斑马鱼幼鱼是一个有吸引力的模型，以研究神经系统疾病的遗传贡献。然而，目前的CRISPR-Cas9方法很难应用于研究行为表型的大型遗传筛选。为了促进快速遗传筛选，我们开发了一种简单的无测序工具来验证gRNA和一种高效的CRISPR-Cas9方法，该方法能够将>90%的注射胚胎直接转化为F0双等位基因敲除。我们证明，F0基因敲除可靠地重演复杂的突变表型，如改变生物钟的分子节律，逃避刺激物的反应，和多参数昼夜运动行为。该技术足够强大，可以敲除同一动物中的多个基因，例如创建透明的三重敲除水晶鱼用于成像。我们的F0敲除方法将斑马鱼从基因到行为表型的实验时间从几个月缩短到一周。
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.