ABSTRACT In animals, early development is dependent on a pool of maternal factors, both RNA and proteins, which are required for basic cellular processes and cell differentiation until zygotic genome activation. The role of the majority of these maternally expressed factors is not fully understood. By exploiting the biallelic editing ability of CRISPR-Cas9, we identify and characterize maternal-effect genes in a single generation, using a maternal crispant technique. We validated the ability to generate biallelic mutations in the germ line by creating maternal crispants that phenocopied previously characterized maternal-effect genes: birc5b, tmi and mid1ip1. Additionally, by targeting maternally expressed genes of unknown function in zebrafish, we identified two maternal-effect zebrafish genes, kpna7 and fhdc3. The genetic identity of these maternal crispants was confirmed by sequencing haploid progeny from F0 females, which allowed the analysis of newly induced lesions in the maternal germ line. Our studies show that maternal crispants allow for the effective identification and primary characterization of maternal-effect genes in a single generation, facilitating the reverse genetics analysis of maternal factors that drive embryonic development.