Efficient CRISPR/Cas9 mutagenesis for neurobehavioral screening in adult zebrafish

Adult zebrafish are increasingly 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. 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, while avoiding the limitations of restriction enzyme-based genotyping. Using a quantifiable behavioral assay, we identify 7 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.