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 (tyrosinase) 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.

Zebrafish larval injections, including subcutaneous and brain-orthotopic tumor xenografting as well as intravenous drug administration, have become central techniques in zebrafish-based research. These procedures, however, remain tedious, technically challenging, and highly dependent on operator skill, limiting throughput and reproducibility. To address these limitations, we introduce an advanced robotic system that automates microinjections into zebrafish larvae, significantly improving efficiency, precision, and reproducibility over manual injections. Here, we provide a detailed protocol for operating the automated injector, a high-throughput system capable of delivering liquids, particles, or cancer cells at three different anatomical sites with superior accuracy and speed compared to trained zebrafish researchers. We outline the essential steps, including setup, calibrations, injection procedures, and troubleshooting, to ensure optimal performance. Furthermore, we provide representative examples of results from using the robot and discuss its potential to facilitate large-scale larval injections, including for drug screens and precision medicine. By integrating an automated injector into the workflows, researchers can overcome limitations of manual injections and accelerate preclinical drug discovery and personalized treatment selection.

This corrects the article 10.3791/68441

Zebrafish larval injections, including subcutaneous and brain-orthotopic tumor xenografting as well as intravenous drug administration, have become central techniques in zebrafish-based research. These procedures, however, remain tedious, technically challenging, and highly dependent on operator skill, limiting throughput and reproducibility. To address these limitations, we introduce an advanced robotic system that automates microinjections into zebrafish larvae, significantly improving efficiency, precision, and reproducibility over manual injections. Here, we provide a detailed protocol for operating the automated injector, a high-throughput system capable of delivering liquids, particles, or cancer cells at three different anatomical sites with superior accuracy and speed compared to trained zebrafish researchers. We outline the essential steps, including setup, calibrations, injection procedures, and troubleshooting, to ensure optimal performance. Furthermore, we provide representative examples of results from using the robot and discuss its potential to facilitate large-scale larval injections, including for drug screens and precision medicine. By integrating an automated injector into the workflows, researchers can overcome limitations of manual injections and accelerate preclinical drug discovery and personalized treatment selection.