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

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.

CRISPR/Cas9 technologies enable a multitude of genomic modifications in cell lines and intact organisms. However, there is a lack of understanding of the molecular DNA repair processes leading to these modifications. This knowledge is urgently needed to be able to edit the genome more precisely to establish crucial molecular and cellular mechanisms operating in an intact organism.

In the first part of the project, we will develop prime editors (PEs), which link the endonuclease Cas9 with a reverse transcriptase, to insert short sequences into the zebrafish genome with high precision. To enable a precise editing process, we will test various PEs and modifications of the guide RNAs and also boost favourable repair mechanisms via chemical compounds such as DNA-PK inhibitors. We will then apply our optimised PE approach to study the Wnt/PCP signalling pathway genes. Wnt genes regulate fundamental processes, including cell migration and polarity during embryogenesis, and we will use our adapted precise genome editing technology to insert short genomic fragments to manipulate the function of the Wnt5b ligand and its cognate receptor Ror2.

To date, the use of PEs has been restricted to catalysing the insertion of short DNA fragments of up to only ca. 30 nucleotides. In the second part of the project, we will seek to overcome this limitation by using an advanced PE to insert larger DNA fragments. This will combine the PE with an integrase. The PE will insert a landing site for an integrase, which will then mediate the recombination of a larger transgene. Using this, we will integrate fluorescent proteins to tag Wnt5b and Ror2 and follow their distribution in the zebrafish.

In this project, we will thus develop precise genome editing methods to alter the genome in a vertebrate in a controlled way, and we will apply this technology to address a fundamental question in embryogenesis as a proof of principle.