Genes and genetic variants associated with human disease are continually being discovered, but validating their causative roles and mechanisms remains a significant challenge. CRISPR/Cas9 genome editing in model organisms like zebrafish can enable phenotypic characterization of founder generation (F0) knockouts (Crispants), but existing approaches are not amenable to high-throughput genetic screening due to high variability, cost, and low phenotype penetrance. To overcome these challenges, here we provide guide RNA (gRNA) selection rules that enable high phenotypic penetrance of up to three simultaneous knockouts in F0 animals following injection of 1-2 gRNAs per gene. We demonstrate a strong transcriptomic overlap in our F0 knockouts and stable knockout lines that take several months to generate. We systematically evaluated this approach across 324 gRNAs targeting 125 genes and demonstrated its utility in studying epistasis, characterizing paralogous genes, and validating human disease gene phenotypes across multiple tissues. Applying our approach in a high-throughput manner, we screened and identified 10 novel neurodevelopmental disorders and 50 hearing genes not previously studied in zebrafish. Altogether, our approach achieves high phenotypic penetrance using low numbers of gRNAs per gene in F0 zebrafish, offering a robust pipeline for rapidly characterizing candidate human disease genes.