Locubiche Sílvia, Ordóñez Víctor, Abad Elena, Scotto di Mase Michele, Di Donato Vincenzo, De Santis Flavia
ZeClinics S.L., Carrer de Laureà Miró, 408-410, 08980 Sant Feliu de Llobregat, Spain.
Faculty of Medicine and Health Sciences, Institute of Neurosciences, University of Barcelona, 08036 Barcelona, Spain.
Int J Mol Sci. 2024 Mar 4;25(5):2991. doi: 10.3390/ijms25052991.
The zebrafish model has emerged as a reference tool for phenotypic drug screening. An increasing number of molecules have been brought from bench to bedside thanks to zebrafish-based assays over the last decade. The high homology between the zebrafish and the human genomes facilitates the generation of zebrafish lines carrying loss-of-function mutations in disease-relevant genes; nonetheless, even using this alternative model, the establishment of isogenic mutant lines requires a long generation time and an elevated number of animals. In this study, we developed a zebrafish-based high-throughput platform for the generation of F0 knock-out (KO) models and the screening of neuroactive compounds. We show that the simultaneous inactivation of a reporter gene () and a second gene of interest allows the phenotypic selection of F0 somatic mutants (crispants) carrying the highest rates of mutations in both loci. As a proof of principle, we targeted genes associated with neurodevelopmental disorders and we efficiently generated de facto F0 mutants in seven genes involved in childhood epilepsy. We employed a high-throughput multiparametric behavioral analysis to characterize the response of these KO models to an epileptogenic stimulus, making it possible to employ kinematic parameters to identify seizure-like events. The combination of these co-injection, screening and phenotyping methods allowed us to generate crispants recapitulating epilepsy features and to test the efficacy of compounds already during the first days post fertilization. Since the strategy can be applied to a wide range of indications, this study paves the ground for high-throughput drug discovery and promotes the use of zebrafish in personalized medicine and neurotoxicity assessment.
斑马鱼模型已成为表型药物筛选的参考工具。在过去十年中，借助基于斑马鱼的检测方法，越来越多的分子从实验室走向了临床应用。斑马鱼和人类基因组之间的高度同源性有助于生成在疾病相关基因中携带功能丧失突变的斑马鱼品系；然而，即使使用这种替代模型，建立同基因突变品系也需要很长的世代时间和大量的动物。在本研究中，我们开发了一个基于斑马鱼的高通量平台，用于生成F0基因敲除（KO）模型和筛选神经活性化合物。我们表明，同时失活该报告基因和第二个感兴趣的基因，可以对在两个位点都携带最高突变率的F0体细胞突变体（crispants）进行表型选择。作为原理验证，我们针对与神经发育障碍相关的基因，成功地在七个与儿童癫痫相关的基因中生成了事实上的F0突变体。我们采用高通量多参数行为分析来表征这些KO模型对致痫刺激的反应，从而能够利用运动学参数来识别癫痫样事件。这些共注射、筛选和表型分析方法的结合，使我们能够生成概括癫痫特征的crispants，并在受精后的头几天就测试化合物的疗效。由于该策略可应用于广泛的适应症，本研究为高通量药物发现奠定了基础，并促进了斑马鱼在个性化医学和神经毒性评估中的应用。
