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

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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.