CRISPR/Cas9 technology of genome editing has greatly facilitated the targeted inactivation of genes in vitro and in vivo in a wide range of organisms. In zebrafish, it allows the rapid generation of knockout lines by simply injecting a guide RNA (gRNA) and Cas9 mRNA into one-cell stage embryos. Here, we report a simple and scalable CRISPR-based vector system for tissue-specific gene inactivation in zebrafish. As proof of principle, we used our vector with the gata1 promoter driving Cas9 expression to silence the urod gene, implicated in heme biosynthesis, specifically in the erythrocytic lineage. Urod targeting yielded red fluorescent erythrocytes in zebrafish embryos, recapitulating the phenotype observed in the yquem mutant. While F0 embryos displayed mosaic gene disruption, the phenotype appeared very penetrant in stable F1 fish. This vector system constitutes a unique tool to spatially control gene knockout and greatly broadens the scope of loss-of-function studies in zebrafish. Display omitted •Urod inactivation using CRISPR yields a fluorescent phenotype in zebrafish embryos•A CRISPR-based vector system enables tissue-specific gene inactivation in zebrafish•The targeting vector can be easily modified for any gene and tissue-specific promoter Ablain et al. describe a vector system for spatial control of gene disruption in zebrafish. Based on the CRISPR technology, this modular vector, which only requires a tissue-specific promoter and a gene-specific target sequence, greatly expands the range of loss-of-function studies in vivo.