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分析脊椎动物发育疾病中基因功能和分子机制的主要模型。正向遗传策略特别有效，已经发现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/或通路在正向筛选中将无法被识别，因此需要反向遗传策略。斑马鱼研究界特别感兴趣的是基因靶向策略的发展，这种策略将允许精确修改基因位点，例如创建模仿人类疾病突变的等位基因，用标记蛋白标记内源基因位点，和/或产生条件等位基因。一种很有前途的反向遗传学方法是利用锌指核酸酶(ZFN)在斑马鱼基因座的精确位置诱导双链断裂(DSB)。诱导的DSB可以通过非同源末端连接(NHEJ)修复，NHEJ通常是突变的，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)修复，使用同源染色体或含有显著同源性的外源供体。我们已经证明，在斑马鱼中，NHEJ介导的ZFN诱导的DSB的修复在体细胞和生殖系中都很容易和有效地发生。在我们的实验中，我们发现，在注射了ZFN编码基因的斑马鱼胚胎中，超过60%的斑马鱼胚胎在成年后携带了新的等位基因，种系频率平均为20%。我们建议在此优化ZFN用于HDR和基因靶向的使用。具体地说，我们建议优化ZFN诱导斑马鱼基因打靶的条件，表征是否可以通过操纵诱导DSB的发育时间或通过操纵调节DSB修复途径选择的成分来增强ZFN介导的HDR，并向斑马鱼群落分发详细的方案和试剂。促进斑马鱼基因打靶的方案和工具的开发将对该领域产生巨大影响。设计精确序列变化的能力，而不是依赖随机突变，将使研究人员能够产生与人类健康和疾病至关重要的等位基因类型，包括条件零等位基因，它允许人们研究特定组织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到感兴趣的基因来研究人类疾病的模型。 公共卫生相关性：脊椎动物模式生物(如斑马鱼)的正向和反向遗传策略，以及随后对突变表型的表型分析，提供了对调节人类发育和疾病的基因、途径和机制的重要洞察。斑马鱼正向遗传策略对于新的基因发现一直很重要，最近，斑马鱼反向遗传策略允许恢复特定感兴趣基因的突变。也就是说，脊椎动物中的定向基因组操作，即对基因序列的精确和特定的修改，在很大程度上局限于小鼠，因为可以在培养中操纵小鼠胚胎干细胞株，并将其重新引入小鼠囊胚。随着设计的锌指核酸酶(ZFN)可以用来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项用于基因打靶的技术，促进携带人类疾病等位基因突变的条件等位基因、标记等位基因或疾病模型的构建，以及许多令人兴奋的可能性。这样的策略将变得至关重要，因为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，并以较晚发病的表型来模拟人类疾病，如行为障碍、神经肌肉疾病和癌症。我们建议开发ZFN介导的基因打靶的优化方案和工具，并将其快速传播到斑马鱼群落。

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.