Conditional gene inactivation is a powerful tool to determine gene function when constitutivemutations result in detrimental effects. The most commonly used technique to achieveconditional gene inactivation employs the Cre/loxP system and its ability to delete DNAsequences flanked by two loxP sites. However, targeting a gene with two loxP sites is timeand labor consuming. Here, we show Cre-Controlled CRISPR (3C) mutagenesis to circumventthese issues. 3C relies on gRNA and Cre-dependent Cas9-GFP expression from the sametransgene. Exogenous or transgenic supply of Cre results in Cas9-GFP expression and subsequent mutagenesis of the gene of interest. The recombined cells become fluorescentlyvisible enabling their isolation and subjection to various omics techniques. Hence, 3Cmutagenesis provides a valuable alternative to the production of loxP-flanked alleles. It mighteven enable the conditional inactivation of multiple genes simultaneously and should beapplicable to other model organisms amenable to single integration transgenesis.