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 critical exons or an entire gene with two loxP sites is time and labor consuming. To circumvent these issues, we developed Cre-Controlled CRISPR (3C) mutagenesis. 3C mutagenesis is simple, fast and allows gene inactivation in a Cre-dependent manner. In contrast to loxP-flanked alleles, the recombined cells become fluorescently visible enabling the isolation of these cells and their subjection to various omics techniques. Moreover, 3C will be scalable and will enable the conditional inactivation of multiple genes simultaneously. Hence, 3C mutagenesis provides a valuable alternative to the production of loxP-flanked alleles and should be applicable to all model organisms amenable to single integration transgenesis.