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

Stefan Hans (), Daniela Zöller, Juliane Hammer, Johanna Stucke, Sandra Spieß, Gokul Kesavan, Volker Kroehne, Juan Sebastian Eguiguren, Diana Ezhkova, Andreas Petzold, Andreas Dahl and Michael Brand ()
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Stefan Hans: Technische Universität Dresden
Daniela Zöller: Technische Universität Dresden
Juliane Hammer: Technische Universität Dresden
Johanna Stucke: Technische Universität Dresden
Sandra Spieß: Technische Universität Dresden
Gokul Kesavan: Technische Universität Dresden
Volker Kroehne: Technische Universität Dresden
Juan Sebastian Eguiguren: Technische Universität Dresden
Diana Ezhkova: Technische Universität Dresden
Andreas Petzold: Technische Universität Dresden
Andreas Dahl: Technische Universität Dresden
Michael Brand: Technische Universität Dresden

Nature Communications, 2021, vol. 12, issue 1, 1-12

Abstract: 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.

Date: 2021
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DOI: 10.1038/s41467-021-21427-6

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