Systems for rapid generation of zebrafish mutants and zebrafish embryo handling

项目摘要 斑马鱼是生物医学研究的重要模式动物。然而，斑马鱼的全部潜力 研究尚未实现，特别是药物发现和大规模模型生成，因为 处理动物和对动物进行基因分型的技术不足。基因分型目前是一个时间，劳动和培训 密集的过程。胚胎必须被抚养到成年，或者必须牺牲胚胎以确定 基因型;突变体很难基因型;和屏幕或药物/治疗试验不能进行 已知基因型的动物，直到年龄较大。最后，基于基因组学进展的高通量技术 编辑技术，如重复的规则间隔短回文重复序列（CRISPR）， 需要人工筛选。Nanonc在过去的一年里带来了第一个活胚胎 基因分型设备ZEG上市（https：//www.example.com），提出了两种新技术的实施方案 使斑马鱼模型生成和用途：通过电穿孔进行基因组编辑的转化; 胚胎处理的自动化。这是因为转基因/诱变和斑马鱼胚胎处理是 与1980年代使用的方法基本相同。突变或转基因是通过 人工注入胚胎。虽然斑马鱼中的CRISPR诱变是高效的， 由于在注射的（G0）动物中存在双等位基因敲低，因此手册要求限制了可以 这限制了下游应用，例如新的转基因系的产生或突变体用于 筛选药物筛选可以在F0幼虫上进行，但需要人类进行注射， 限制了可以使用的动物数量。另一个主要问题是胚胎的处理 通过手动移液器转移，例如，转移到96孔板中，这可能需要单个使用者每 一次移动一个胚胎。为了解决这些问题，我们建议开发两种产品， 将与市售的ZEG产品整合：首先，开发一种用于高- 在斑马鱼（"Zapper"）中的高通量CRISPR诱变和转基因。电穿孔技术具有 在概念验证实验中，已经显示出能够将分子构建体传递到斑马鱼胚胎中，但是 尚未测试CRISPR诱变/转基因或可扩展性。我们将测试、开发和实施 一种用于将构建体递送至斑马鱼胚胎的基于电穿孔的系统。第二，我们会发展一个 用于快速装载胚胎的斑马鱼胚胎处理系统（"Zipper"）。药物或突变体筛选在96- 或324孔板，或ZEG（斑马鱼胚胎基因分型）设备，需要费力的手动加载/卸载 斑马鱼的胚胎。机器人选项的成本超过100，000美元，并且难以解决问题或互换 使用之间。我们用于快速分配斑马鱼胚胎的低成本机械设备是新颖的，可申请专利， 并将在学术界和工业界的实验室中立即使用斑马鱼。

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.