Efficient CRISPR/Cas9 mutagenesis for neurobehavioral screening in adult zebrafish

Adult zebrafish are a premier vertebrate model to interrogate mechanisms of disease development and tissue regeneration. In this issue, Shaw and Mokalled used high efficiency genome editing tools to introduce 28 mutations in 17 genes with efficiencies exceeding 85%. A quantifiable swim endurance test identified 7 mutations with reduced functional recovery after spinal cord injury. This study provides an experimental pipeline to perform medium- to large-scale neurobehavioral screens in adult zebrafish.Abstract Adult zebrafish are widely used to interrogate mechanisms of disease development and tissue regeneration. Yet, the prospect of large-scale genetics in adult zebrafish has traditionally faced a host of biological and technical challenges, including inaccessibility of adult tissues to high-throughput phenotyping and the spatial and technical demands of adult husbandry. Here, we describe an experimental pipeline that combines high-efficiency CRISPR/Cas9 mutagenesis with functional phenotypic screening to identify genes required for spinal cord repair in adult zebrafish. Using CRISPR/Cas9 dual-guide ribonucleic proteins, we show selective and combinatorial mutagenesis of 17 genes at 28 target sites with efficiencies exceeding 85% in adult F0 “crispants”. We find that capillary electrophoresis is a reliable method to measure indel frequencies. Using a quantifiable behavioral assay, we identify seven single- or duplicate-gene crispants with reduced functional recovery after spinal cord injury. To rule out off-target effects, we generate germline mutations that recapitulate the crispant regeneration phenotypes. This study provides a platform that combines high-efficiency somatic mutagenesis with a functional phenotypic readout to perform medium- to large-scale genetic studies in adult zebrafish.