Precise base editing for the in vivo study of developmental signaling and human pathologies in zebrafish.

Marion Rosello,Shahad Albadri,Marina C Mione,Jean-Paul Concordet,François Czarny,Filippo Del Bene,Juliette Vougny

doi:10.7554/elife.65552

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 protospacer adjacent motif, expanding the base editing possibilities in zebrafish. Using these approaches, we first generated a base change in the ctnnb1 gene, mimicking oncogenic an mutation 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.

Highlights

With the recent technological advances in precise gene editing, the use of zebrafish in genetic engineering studies has drastically increased in the last 5 years (Patton and Tobin, 2019; Santoriello and Zon, 2012)
The CRISPR/ Cas9 system is a remarkably powerful gene-editing tool (Sander and Joung, 2014) that enables the rapid and efficient generation of loss-of-function mutations in this animal model. This system relies on the specific binding of a sgRNA-Cas9 complex that initially interacts with DNA 20 base pair upstream of a NGG protospacer adjacent motif (PAM) sequence that triggers the Cas9 protein to introduce a double-strand break (DSB)
BE4-gam base editing for the endogenous activation of Wnt signaling pathway

Summary

Introduction

With the recent technological advances in precise gene editing, the use of zebrafish in genetic engineering studies has drastically increased in the last 5 years (Patton and Tobin, 2019; Santoriello and Zon, 2012). The CRISPR (clustered regularly interspaced short palindromic repeats)/ Cas system is a remarkably powerful gene-editing tool (Sander and Joung, 2014) that enables the rapid and efficient generation of loss-of-function mutations in this animal model. This system relies on the specific binding of a sgRNA-Cas complex that initially interacts with DNA 20 base pair (bp) upstream of a NGG protospacer adjacent motif (PAM) sequence that triggers the Cas protein to introduce a double-strand break (DSB). A CBE was shown to work but with limited efficiencies, inducing less than 29% of gene editing and, in most

Methods

Results

Conclusion