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

Abstract

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 Parkinson's 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.

Unbiased intestinal single cell transcriptomics reveals previously uncharacterized enteric nervous system populations in larval zebrafish

“…One of the vertebrate animal models that is regularly used to study ENS development, is the zebrafish (4). Zebrafish are highly suitable for genetic manipulation, develop rapidly, ex-utero and are transparent, which makes them extremely valuable for screening novel disease genes and tracing developmental processes (5, 6). However, the precise composition and specification of different neuronal and glial subtypes in the zebrafish ENS, remains unclear.…”

A targeted CRISPR-Cas9 mediated F0 screen identifies genes involved in establishment of the enteric nervous system

“…Similar to our methodology, they utilized the Tg(-8.3phox2bb:Kaede) transgenic zebrafish (Harrison et al, 2014) and performed a comparative analysis between morpholino-mediated knockdown and CRISPR knockouts of six genes. Another recent ENS F0 screen was able to identify the role of ten transcription factors, finding alterations in the number of ENs, and in gut motility using Tg(phox2bb:GFP) embryos (Davidson et al, 2021). One of the features of our screen was the capability of identifying most of our candidate genes with alterations in the ENS.…”

A targeted CRISPR-Cas9 mediated F0 screen identifies genes involved in establishment of the enteric nervous system

“…Similar to our methodology, they utilized the Tg(-8.3phox2bb:Kaede) transgenic zebrafish [18] and performed a comparative analysis between morpholino-mediated knockdown and CRISPR knockouts of six genes. Another recent ENS F0 screen identified the role of ten transcription factors, finding alterations in the number of ENs and gut motility using Tg(phox2bb:GFP) embryos [50]. A few limitations have been known to occur and vary widely in the generation of crispants models, such as phenotype penetrance, mutagenesis efficiencies, and somatic mosaicism [51].…”