A rapid F0 CRISPR screen in zebrafish to identify regulators of neuronal development in the enteric nervous system

The enteric nervous system (ENS) provides the intrinsic innervation of the gastrointestinal (GI) tract with millions of neurons and diverse neuronal subtypes and glial cells. The ENS regulates essential gut functions such as motility, nutrient uptake, and immune response, but basic information about the genes that control ENS neuronal specification and differentiation remains largely unknown. Deficits in ENS neuron numbers and composition cause gut dysfunction with debilitating GI symptoms, and are associated with e.g. Hirschsprung disease, inflammatory gut diseases, autism spectrum disorder, and neurodegenerative diseases such as Parkinsons disease. The genetic basis of most of these ENS disorders remains unknown. Recent transcriptomic analyses have identified many candidate genes for regulating ENS neurogenesis. However, functional evaluation of these candidate genes significantly lags because experimental testing of their role in ENS neurogenesis is time-consuming and expensive. Here, we have developed a rapid, scalable F0 CRISPR genome editing screen in zebrafish to functionally determine which candidate genes control neuronal development in the ENS. Proof-of-concept experiments targeting the known ENS regulators sox10 and ret phenocopy stable mutants with high efficiency and precision showing that our approach is reliable to identify regulators ENS neurogenesis using F0 guide RNA-injected larvae (F0 crispants). We then evaluate the role of 10 transcription factor genes for regulating ENS neurogenesis and function. Pools of guide RNAs targeting 2-3 candidate genes are co-injected with Cas9 protein into one-cell stage phox2bb:GFP transgenic zebrafish embryos to directly assess qualitative change in ENS neuron numbers compared to controls in 6-day old F0 crispants. Target genes from crispant pools exhibiting reduced ENS neuronal numbers were then tested individually to identify the responsible gene(s). We identify five transcription factors that show a reduction in ENS neurons indicating an influence on enteric progenitor cell differentiation into ENS neurons. Adding a simple and efficient test to further assess crispant gut motility, we find that loss-of-function of two of the transcription factor genes reduced intestinal transit of fluorescently labeled food through the gut. In summary, our novel, multistep, yet straight-forward CRISPR screening approach in zebrafish enables testing the genetic basis of ENS developmental and disease gene functions that will facilitate high-throughput evaluation of the manifold candidate genes emerging from transcriptomic, genome-wide association or other ENS-omics studies. Such in vivo ENS crispant screens will contribute to a better understanding of ENS neuronal development regulation in vertebrates and what goes awry in ENS disorders.