Targeted genome modification in zebrafish using zinc finger nucleases

描述（申请人提供）：斑马鱼模型系统已成为研究脊椎动物发育疾病过程中基因功能和分子机制的主要模型。前瞻性遗传策略特别强大，已经确定了许多与发育和疾病有关的基因。然而，由于各种原因，一些关键基因和/或途径将无法在正向筛选中鉴定，从而需要反向遗传策略。斑马鱼研究界特别感兴趣的是基因靶向策略的开发，该策略将允许基因位点的精确修饰，例如，产生模拟人类疾病突变的等位基因，用标记蛋白标记内源基因座，和/或产生条件等位基因。利用锌指核酸酶（ZFN）在斑马鱼基因座内的精确位置诱导双链断裂（DSB）是一种很有前途的新的反向遗传学方法。诱导的DSB可以通过非同源末端连接（NHEJ）修复，这通常是诱变性的，在DSB位点产生小的插入和缺失。或者，DSB可以通过同源定向修复（HDR）来修复，使用同源染色体或含有显著同源性的外源提供的供体。我们已经表明，在斑马鱼中，NHEJ介导的ZFN诱导的DSB的修复在体细胞和种系中容易且有效地发生。在我们的实验中，我们表明，超过60%的斑马鱼胚胎注射ZFN编码mRNA，并生长到成年携带新的等位基因，生殖系频率平均为20%。我们在这里建议优化ZFN用于HDR和基因靶向的使用。具体而言，我们建议优化ZFN诱导的斑马鱼基因靶向的条件，以表征ZFN介导的HDR是否可以通过操纵DSB诱导的发育时机或通过操纵调节DSB修复途径选择的组件来增强，并将详细的方案和试剂分发给斑马鱼社区。促进斑马鱼基因靶向的方案和工具的开发将对该领域产生巨大影响。工程精确序列变化的能力，而不是依赖于随机诱变，将使研究人员能够产生与人类健康和疾病密切相关的等位基因类型，包括条件无效等位基因，这使得人们能够研究特定组织中或特定时间的基因功能，这些基因功能通常由于多效性功能而被掩盖，以及通过将特定的人类突变工程化到感兴趣的基因中来建立人类疾病模型。 公共卫生相关性：脊椎动物模式生物（如斑马鱼）中的正向和反向遗传策略以及随后的突变表型表型分析为调节人类发育和疾病的基因、途径和机制提供了重要的见解。斑马鱼正向遗传策略对于新基因的发现非常重要，最近，斑马鱼反向遗传策略已经允许在特定的感兴趣基因中恢复突变。也就是说，脊椎动物中的靶向基因组操作，即基因序列的精确和特异性修饰，在很大程度上仅限于小鼠，这是由于可以在培养中操作并重新引入小鼠囊胚的小鼠胚胎干细胞系的可用性。随着设计的锌指核酸酶（ZFN）可用于在斑马鱼基因组的精确位置产生双链断裂的发现，我们现在准备优化这种基因靶向技术，促进条件等位基因，标记等位基因或携带人类疾病等位基因突变的疾病模型的构建，以及许多其他令人兴奋的可能性。当我们开始剖析在多个时间和多个组织中控制发育过程的信号通路，并模拟具有迟发型表型的人类疾病时，这些策略将变得至关重要，如行为障碍、神经肌肉疾病和癌症。我们在这里建议开发优化ZFN介导的基因靶向的协议和工具，并迅速传播到斑马鱼社区。

DESCRIPTION (provided by applicant): The zebrafish model system has become a main model used to dissect gene function and molecular mechanisms during vertebrate development disease. Forward genetic strategies have been particularly powerful and have identified many genes involved in development and disease. However, for a variety of reasons, some critical genes and/or pathways will not be identified in forward screens, necessitating the need for reverse genetic strategies. Of particular interest to the zebrafish research community is the development of gene targeting strategies that will allow the precise modification of a gene locus, e.g., to create an allele mimicking a human disease mutation, tag the endogeneous locus with a marker protein, and/or to generate conditional alleles. A promising new reverse genetics approach is the use of zinc finger nucleases (ZFNs) to induce double strand breaks (DSBs) at precise positions within zebrafish loci. Induced DSBs can be repaired via non-homologous end joining (NHEJ), which is often mutagenic, creating small insertions and deletions at the DSB site. Alternatively, DSBs can be repaired by homology directed repair (HDR), using either the homologous chromosome or an exogenously supplied donor containing significant homology. We have shown that in zebrafish, NHEJ-mediated repair of ZFN-induced DSBs occurs readily and efficiently in both somatic cells and the germline. In our experiments, we show that over 60% of zebrafish embryos injected with ZFN-encoding mRNA and grow to adulthood carry new alleles at germline frequencies averaging 20%. We propose here to optimize the use of ZFNs for HDR and gene targeting. Specifically, we propose to optimize conditions for ZFN-induced gene targeting in zebrafish, to characterize whether ZFN-mediated HDR can be enhanced by manipulating the developmental timing at which the DSBs are induced or by manipulating components that regulate DSB repair pathway choice, and to distribute detailed protocols and reagents to the zebrafish community. Development of protocols and tools that facilitate gene targeting in zebrafish will have a huge impact on the field. The ability to engineer precise sequence changes, instead of relying on random mutagenesis, will allow researchers to generate the types of alleles that are critically relevant to human health and disease, including conditional null alleles, which allow one to study gene function in specific tissues or at specific times that is normally masked due to pleiotropic function, and models of human disease by engineering specific human mutations into the gene of interest. PUBLIC HEALTH RELEVANCE: Forward and reverse genetic strategies in vertebrate model organisms, such as the zebrafish, and the subsequent phenotypic analysis of mutant phenotypes, has provided critical insight into genes, pathways, and mechanisms regulating human development and disease. Zebrafish forward genetic strategies have been important for new gene discovery, and more recently, zebrafish reverse genetic strategies have allowed recovery of mutations in specific genes of interest. That said, targeted genome manipulation in vertebrates, the precise and specific modification of gene sequence, has been largely restricted to the mouse, due to the availability of mouse embryonic stem cell lines that can be manipulated in culture and reintroduced into mouse blastocysts. With the discovery that designed zinc finger nucleases (ZFNs) can be used to generate double-strand breaks at precise positions in the zebrafish genome, we are now poised to optimize this technology for gene targeting, facilitating the construction of conditional alleles, tagged alleles, or disease models carrying human disease allele mutations, among many other exciting possibilities. Such strategies will become critically important as we begin to dissect signaling pathways that control developmental processes at multiple times and in multiple tissues and to model human diseases with later-onset phenotypes, as wide-ranging as behavioral disorders, neuromuscular disease, and cancer. We propose here to develop protocols and tools for optimization of ZFN-mediated gene targeting and rapidly disseminate them to the zebrafish community.