Establishing precise genome editing in zebrafish and its application to advance understanding of the Wnt/PCP signalling pathway

基因组及其组成基因由双链DNA组成，双链DNA可以被环境因素无意地或有意地破坏，使用称为核酸酶的蛋白质。细胞已经开发出修复这些双链断裂的机制，这些机制可以重现原始序列，但也会产生DNA改变。最近，CRISPR/Cas9系统已被用于在DNA中产生断裂并应用于修饰DNA序列。然而，基本的修复机制还没有很好地理解，使其使用不可预测的许多应用程序。了解这些修复机制如何运作是非常必要的，以允许准确的基因组编辑。最近，已经开发了主要编辑器，其联合收割机两种酶组分，即内切核酸酶Cas9和工程逆转录酶，允许在DNA断裂内精确安装小编辑。我们的工业合作伙伴阿斯利康（AstraZeneca）已经证明，这些主要编辑器可以诱导DNA修复机制，以精确编辑基因组序列并在组织培养中插入外源DNA。然而，在复杂的脊椎动物中调节这些修饰的机制的应用和理解尚未建立。这些知识将是至关重要的，使我们能够解决许多基本问题，在胚胎发育和成年组织在脊椎动物，特别是在了解细胞信号系统。通过在斑马鱼基因组中的特定位点诱导这些DNA断裂并触发特定的DNA修复机制，我们提出在脊椎动物体内开发更精确的编辑过程。在本申请中，我们将研究胚胎发生中的细胞间通讯，这是确定细胞类型多样性的基础，从而形成组织和器官，甚至整个生物体。一组细胞产生的信号如何通过浓度梯度传递到邻近组织中的细胞，以协调它们的行为，在完整的脊椎动物中从未被可视化。我们将应用本提案中开发的精确编辑方法，首次可视化Wnt/PCP信号如何形成梯度，以影响斑马鱼胚胎中复杂的细胞迁移。为了准备这项提案，我们已经启动了在斑马鱼中开发精确基因组编辑的过程，并开始生成可能允许将特定核苷酸插入DNA断裂的主要编辑器。我们将使用这些遗传工具来高精度地改变基因组密码，以解决Wnt/PCP配体与其受体的相互作用如何影响梯度形成。然后，我们将扩展我们的工具集，使用先进的prime编辑器进行精确的基因组编辑，以插入更大的DNA链。最后，我们将用它来连接Wnt配体和它们的受体与荧光蛋白，使我们能够确定在发育中的斑马鱼组织中配体和受体的局部浓度和位置。这将使我们能够第一次在活体脊椎动物中绘制Wnt/PCP梯度。该项目将大大扩展我们对斑马鱼精确基因组编辑的了解，并为研究界提供准确的基因组编辑工具。我们还将提供这些工具的原理证明，以首次在脊椎动物中可视化内源性Wnt/PCP信号梯度。

Genomes and their comprising genes are made of double-stranded DNA, which can be broken unintentionally by environmental factors or intentionally, using proteins called nucleases. Cells have developed mechanisms to repair these double-strand breaks that can recapitulate the original sequence but also generate DNA alterations. Recently, the CRISPR/Cas9 system has been used to generate breaks in the DNA and applied to modify DNA sequences. However, the underlying repair mechanisms are not well understood to date, making its use unpredictable for many applications. Understanding how these repair mechanisms operate is much needed to allow for accurate genome editing. Recently, prime editors have been developed, which combine two enzymatic components, namely an endonuclease, Cas9 and an engineered reverse transcriptase, allowing the accurate installation of small edits within the DNA break. Our industrial partner, AstraZeneca, has shown that these prime editors can induce DNA repair mechanisms to precisely edit genomic sequences and insert exogenous DNA in tissue culture. However, the application, and understanding of the mechanisms regulating these modifications, in a complex vertebrate have not been established. This knowledge will be critically important in allowing us to address many fundamental questions in both embryogenesis and adult tissues in vertebrates, notably in understanding cell signalling systems. Through inducing these DNA breaks at specific sites in the zebrafish genome and triggering particular DNA repair mechanisms, we propose to develop a more precise editing process in vertebrates in vivo. Within this application, we will investigate cell-to-cell communication in embryogenesis which is fundamental to determining cell-type diversity and, thus, forming tissues and organs, and indeed the entire organism. How the signals produced by one group of cells are relayed through concentration gradients to cells in neighbouring tissues to orchestrate their behaviour has never been visualised in an intact vertebrate. We will apply the precision editing methods developed in this proposal to visualise for the first time how Wnt/PCP signals form a gradient to influence complex cell migration in the zebrafish embryo.In preparation for this proposal, we have initiated the process for developing precise genome editing in the zebrafish and begun to generate prime editors that potentially allow for the insertion of specific nucleotides into a DNA break. We will use these genetic tools to change the genomic code with high precision to address how the interaction of the Wnt/PCP ligands with their receptors influences gradient formation. We will then expand our toolset for precise genome editing with an advanced prime editor to insert larger DNA strands. Finally, we will use this to link Wnt ligands and their receptors with fluorescent proteins enabling us to determine the local concentration and position of ligands and receptors in the developing zebrafish tissue. This will allow us to map the Wnt/PCP gradient in a living vertebrate animal for the first time. This project will significantly expand our knowledge of precise genome editing in zebrafish and provide accurate genomic editing tools for the research community. We will also provide a proof-of-principle of these tools to visualise the endogenous Wnt/PCP signalling gradient for the first time in vertebrates.