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脊椎动物模式生物。然而，斑马鱼的全部潜力 研究尚未实现，特别是在药物发现和大规模模型生成方面，因为 没有足够的技术来处理动物和对动物进行基因分型。目前，基因分型是一项时间、劳动和培训 密集的过程。胚胎必须被培养到成年，或者必须牺牲胚胎才能确定 基因类型；突变很难进行基因分型；不能进行筛查或药物/治疗试验 已知基因的动物，直到更老的年龄。最后，基于基因组技术进展的高通量技术 诸如聚类规则间隔短回文重复(CRISPR)之类的编辑技术受到 人工筛查的要求。在过去的一年里，Nanonc带来了第一个此类活胚胎 基因分型设备ZEG推向市场(https://www.wfluidx.com)，建议实施两项新技术 增强斑马鱼模型的生成和使用能力：通过电穿孔转变基因组编辑；以及 胚胎处理的自动化。这是因为转基因/突变和斑马鱼胚胎处理是 在方法上与1980年代使用的方法基本相同。突变，或转基因，是由 人工注射到胚胎中。而在斑马鱼中进行CRISPR突变是高效的，通常可以实现 双等位基因敲除在注射的(G0)动物中，手动要求限制了可以 这限制了下游的应用，例如产生新的转基因品系或使用突变体 放映。药物筛选可以在F0幼虫身上进行，但要求有人类进行注射 限制可以使用的动物数量。另一个主要问题是对胚胎的处理 例如，通过手动吸移管转移到96孔板中，这可能需要单个用户将高达30‘ 通过一次移动一个胚胎来进行盘状培养。为了解决这些问题，我们建议开发两种产品， 将与市售的ZEG产品相结合：首先，开发一种用于高频电穿孔的系统。 斑马鱼(‘Zapper’)的吞吐量CRISPR突变和转基因。电穿孔技术已经 在概念验证实验中被证明能够将分子结构输送到斑马鱼胚胎，但 尚未对CRISPR突变/转基因或可扩展性进行测试。我们将测试、开发和实现 一种基于电穿孔的斑马鱼胚胎构建系统。第二，我们将制定一项 斑马鱼胚胎处理系统用于快速加载胚胎(‘Zipper’)。在96-年进行药物或突变筛查- 或324孔板，或ZEG(斑马鱼胚胎基因分型)装置，需要费力的人工装卸 斑马鱼胚胎。机器人选件的成本超过10万美元，而且很难排除故障或互换 在两种用途之间。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、可申请专利的、 并将立即在学术界和工业部门的斑马鱼实验室中使用。

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.