Cre-Controlled CRISPR mutagenesis provides fast and easy conditional gene inactivation in zebrafish

Stefan Hans,Daniela Zöller,Michael Brand,Juan Sebastian Eguiguren,Diana Ezhkova,Volker Kroehne,Juliane Hammer,Sandra Spieß,Gokul Kesavan,Johanna Stucke,Andreas Petzold,Andreas Dahl

doi:10.1038/s41467-021-21427-6

Stefan Hans, Daniela Zöller + Show 10 more

Open Access

https://doi.org/10.1038/s41467-021-21427-6

Copy DOI

Abstract

Conditional gene inactivation is a powerful tool to determine gene function when constitutive mutations result in detrimental effects. The most commonly used technique to achieve conditional gene inactivation employs the Cre/loxP system and its ability to delete DNA sequences flanked by two loxP sites. However, targeting a gene with two loxP sites is time and labor consuming. Here, we show Cre-Controlled CRISPR (3C) mutagenesis to circumvent these issues. 3C relies on gRNA and Cre-dependent Cas9-GFP expression from the same transgene. Exogenous or transgenic supply of Cre results in Cas9-GFP expression and subsequent mutagenesis of the gene of interest. The recombined cells become fluorescently visible enabling their isolation and subjection to various omics techniques. Hence, 3C mutagenesis provides a valuable alternative to the production of loxP-flanked alleles. It might even enable the conditional inactivation of multiple genes simultaneously and should be applicable to other model organisms amenable to single integration transgenesis.

Highlights

Conditional gene inactivation is a powerful tool to determine gene function when constitutive mutations result in detrimental effects
Reverse genetics is the dominant approach, building on precise gene targeting via homologous recombination in embryonic stem cells which has revolutionized the study of mammalian biology and human medicine[5]
Previous reports showed that transgenic zebrafish with stable Cas[9] and gRNA expression can be used to induce biallelic gene inactivation in somatic cells[21,30]

Summary

Introduction

Conditional gene inactivation is a powerful tool to determine gene function when constitutive mutations result in detrimental effects. The most commonly used technique to achieve conditional gene inactivation employs the Cre/loxP system and its ability to delete DNA sequences flanked by two loxP sites. 3C mutagenesis provides a valuable alternative to the production of loxP-flanked alleles It might even enable the conditional inactivation of multiple genes simultaneously and should be applicable to other model organisms amenable to single integration transgenesis. Conditional gene inactivation can be achieved in a Cre-dependent manner if a gene or critical exon is flanked by loxP sites (floxed). CRISPR (3C) mutagenesis as an easy and straightforward system that allows conditional gene inactivation in a Cre-dependent manner. Following Cre-mediated recombination and expression of Cas9-GFP, presumptive mutant cells become fluorescently visible which enables the isolation of these cells and their subjection to various downstream omics techniques, like transcriptomics. 3C mutagenesis provides a valuable alternative to the production of floxed alleles and has the potential for applications in other model organisms amenable to single integration transgenesis

Methods

Results

Conclusion