Abstract
Base editing is a powerful CRISPR-based technology for introducing precise substitutions into the genome. This technology greatly advances mutagenesis possibilities in vivo, particularly in zebrafish, for which the generation of precise point mutations is still challenging. Zebrafish have emerged as an important model for genetic studies and in vivo disease modeling. With the development of different base editor variants that recognize protospacer-adjacent motifs (PAMs) other than the classical 5′-NGG-3′ PAM, it is now possible to design and test several guide RNAs to find the most efficient way to precisely introduce the desired substitution. Here, we describe the experimental design strategies and protocols for cytosine base editing in zebrafish, from guide RNA design and selection of base editor variants to generation of the zebrafish mutant line carrying the substitution of interest. By using co-selection by introducing a loss-of-function mutation in genes necessary for the formation of pigments, injected embryos with highly efficient base editing can be directly analyzed to determine the phenotypic impact of the targeted substitution. The generation of mutant embryos after base editor injections in zebrafish can be completed within 2 weeks.
Key points
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Cytosine base editing can introduce specific C-to-T transitions in the genome. This protocol for cytosine base editing in zebrafish covers guide RNA design, selection of base editors and generation of mutant lines carrying the substitution of interest.
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Compared with standard CRISPR–Cas9-based approaches, base editors generate precise substitutions without double-strand DNA breaks, improving efficiency over previous methods to generate zebrafish lines with precise human pathological mutations for in vivo disease modeling.
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Acknowledgements
We thank C. Giovannangeli and A. Miccio for helpful discussions and sharing plasmids. M.R. was supported by the Fondation pour la Recherche Médicale (FRM grant number ECO20170637481) and la Ligue Nationale Contre le Cancer. This work was supported by ANR-18-CE16 ‘iReelAx’, ANR-20-CE17-0020-02 ‘INCEPTION’, ANR-11-INBS-0014-TEFOR, UNADEV in partnership with ITMO NNP/AVIESAN (national alliance for life sciences and health, UNADEV-19UU51-DEL BENE), Fondation pour la Recherche Médicale (MND202003011485), in the framework of research on vision and IHU FOReSIGHT (ANR-18-IAHU-0001) supported by French state funds managed by the Agence Nationale de la Recherche within the Investissements d’Avenir program.
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All the authors contributed to the protocol development. M.R. and M.S. did the experimental work. M.R. and F.D.B. wrote the manuscript with input and editing from J.-P.C. and M.S.
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Key references using this protocol
Rosello, M. et al. Nat. Commun. 13, 3435 (2022): https://doi.org/10.1038/s41467-022-31172-z
Rosello, M. et al. eLlife 10, e65552 (2021): https://doi.org/10.7554/eLife.65552
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Rosello, M., Serafini, M., Concordet, JP. et al. Precise mutagenesis in zebrafish using cytosine base editors. Nat Protoc 18, 2794–2813 (2023). https://doi.org/10.1038/s41596-023-00854-3
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DOI: https://doi.org/10.1038/s41596-023-00854-3
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