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


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.

SUBMITTER: Hans S 

PROVIDER: S-EPMC7893016 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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

Hans Stefan S   Zöller Daniela D   Hammer Juliane J   Stucke Johanna J   Spieß Sandra S   Kesavan Gokul G   Kroehne Volker V   Eguiguren Juan Sebastian JS   Ezhkova Diana D   Petzold Andreas A   Dahl Andreas A   Brand Michael M  

Nature communications 20210218 1


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  ...[more]

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