Systems for rapid generation of zebrafish mutants and zebrafish embryo handling

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Project Summary Zebrafish is an important vertebrate model organism for biomedical research. However, the full potential of zebrafish research has not been realized, in particular for drug discovery and for large-scale model generation, because of insufficient technologies to handle and genotype animals. Genotyping currently is a time, labor, and training intensive process. Embryos must either be raised to adulthood or embryos must be sacrificed to determine genotypes; mutants are difficult to genotype; and screens or drug/therapeutics trials cannot be performed on animals of known genotype until an older age. Finally, high-throughput technologies based on advances in genomic editing technology such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) are limited by a requirement for manual screening. Nanonc, which in the past year has brought the first-of-its-kind live embryo genotyping device ZEG to market (https://www.wfluidx.com), proposes implementation of two novel technologies to empower zebrafish model generation and use: the transformation of genome-editing via electroporation; and the automation of embryo handling. This is because transgenesis/mutagenesis and zebrafish embryo handling are essentially identical in approach to methods used in the 1980s. Mutagenesis, or transgenesis, are performed by manual injection into embryos. While CRISPR mutagenesis in zebrafish is highly efficient and typically achieves bi-allelic knock-down in the injected (G0) animal, the manual requirement limits the total number of animals that can be generated, which limits downstream applications such as new transgenic line generation or use of mutants for screening. Drug screens could be performed on F0 larvae, but the requirement to have humans do the injection limits the number of animals that can be used. The other major problem is that handling of embryos is performed by manual pipette transfer, for example, into 96-well plates, that can require a single user to dedicate up to 30’ per plate by moving embryos one at a time. To solve these problems, we propose the development of two products that will integrate with the commercially available ZEG product: First, developing an electroporation system for high- throughput CRISPR mutagenesis and transgenesis in zebrafish (‘Zapper’). Electroporation techniques have been shown capable of delivering molecular constructs to zebrafish embryos in proof-of-concept experiments, but have not been tested for CRISPR mutagenesis/transgenesis or for scalability. We will test, develop, and implement an electroporation-based system for delivery of constructs to zebrafish embryos. Second, we will develop a zebrafish embryo handling system for rapid loading of embryos (‘Zipper’). Drug or mutant screening in 96- or 324-well plates, or the ZEG (Zebrafish Embryo Genotyping) device, require laborious manual loading/unloading of zebrafish embryos. Robotic options cost in excess of $100,000 and are difficult to trouble-shoot or to interchange between uses. Our lower-cost mechanical device for the rapid dispensing of zebrafish embryos is novel, patentable, and would find immediate use in labs working with zebrafish in both academia and industry sectors.