Precise base editing for the in vivo study of developmental signaling and human pathologies in zebrafish
- Institut de la Vision, France
- Institut Curie, France
- University of Trento, Italy
- Muséum National d'Histoire Naturelle, France
Abstract
While zebrafish is emerging as a new model system to study human diseases, an efficient methodology to generate precise point mutations at high efficiency is still lacking. Here we show that base editors can generate C-to-T point mutations with high efficiencies without other unwanted on-target mutations. In addition, we established a new editor variant recognizing an NAA PAM, expanding the base editing possibilities in zebrafish. Using these approaches, we first generated a base change in the ctnnb1 gene, mimicking oncogenic mutations of the human gene known to result in constitutive activation of endogenous Wnt signaling. Additionally, we precisely targeted several cancer-associated genes including cbl. With this last target we created a new zebrafish dwarfism model. Together our findings expand the potential of zebrafish as a model system allowing new approaches for the endogenous modulation of cell signaling pathways and the generation of precise models of human genetic disease associated-mutations.
Data availability
All data generated or analysed during this study are included in the manuscript and supporting files.
Article and author information
Author details
Funding
Agence Nationale de la Recherche (ANR-18-CE16 iReelAx"")
- Filippo Del Bene
Agence Nationale de la Recherche ([ANR-18-IAHU-0001)
- Filippo Del Bene
Fondation pour la Recherche Médicale (ECO20170637481)
- Marion Rosello
Ligue Nationale Contre le Cancer
- Marion Rosello
UNADEV/AVIESAN
- Filippo Del Bene
World Wide Cancer Research (grant no. 0624)
- Marina C Mione
LILT -Trento (Program 5 per mille)
- Marina C Mione
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: All procedures were performed on zebrafish embryos in accordance with the European Communities Council Directive (2010/63/EU) and French law (87/848) and approved by the Sorbonne Université ethic committee (Charles Darwin) and the French Ministry for research (APAFIS agreement #21323 2019062416186982) and by the Institut Curie ethic committee and the French Ministry for research (APAFIS agreement #6031 2016070822342309)
Copyright
© 2021, Rosello et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Precise base editing for the in vivo study of developmental signaling and human pathologies in zebrafish
eLife 10:e65552.
https://doi.org/10.7554/eLife.65552
Further reading
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- Developmental Biology
During the first lineage segregation, mammalian embryos generate the inner cell mass (ICM) and trophectoderm (TE). ICM gives rise to the epiblast (EPI) that forms all cell types of the body, an ability referred to as pluripotency. The molecular mechanisms that induce pluripotency in embryos remain incompletely elucidated. Using knockout (KO) mouse models in conjunction with low-input ATAC-seq and RNA-seq, we found that Oct4 and Sox2 gradually come into play in the early ICM, coinciding with the initiation of Sox2 expression. Oct4 and Sox2 activate the pluripotency-related genes through the putative OCT-SOX enhancers in the early ICM. Furthermore, we observed a substantial reorganization of chromatin landscape and transcriptome from the morula to the early ICM stages, which was partially driven by Oct4 and Sox2, highlighting their pivotal role in promoting the developmental trajectory toward the ICM. Our study provides new insights into the establishment of the pluripotency network in mouse preimplantation embryos.
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