CRISPR gRNA phenotypic screening in zebrafish reveals pro-regenerative genes in spinal cord injury

Abstract

Zebrafish exhibit robust regeneration following spinal cord injury, promoted by macrophages that control post-injury inflammation. However, the mechanistic basis of how macrophages regulate regeneration is poorly understood. To address this gap in understanding, we conducted a rapid in vivo phenotypic screen for macrophage-related genes that promote regeneration after spinal injury. We used acute injection of synthetic RNA Oligo CRISPR guide RNAs (sCrRNAs) that were pre-screened for high activity in vivo. Pre-screening of over 350 sCrRNAs allowed us to rapidly identify highly active sCrRNAs (up to half, abbreviated as haCRs) and to effectively target 30 potentially macrophage-related genes. Disruption of 10 of these genes impaired axonal regeneration following spinal cord injury. We selected 5 genes for further analysis and generated stable mutants using haCRs. Four of these mutants ( tgfb1a, tgfb3, tnfa, sparc) retained the acute haCR phenotype, validating the approach. Mechanistically, tgfb1a haCR-injected and stable mutant zebrafish fail to resolve post-injury inflammation, indicated by prolonged presence of neutrophils and increased levels of il1b expression. Inhibition of Il-1β rescues the impaired axon regeneration in the tgfb1a mutant. Hence, our rapid and scalable screening approach has identified functional regulators of spinal cord regeneration, but can be applied to any biological function of interest.

Author summary

Nerve connections that are severed in spinal cord injury do not heal, which can lead to permanent paralysis. Lack of repair may in part be due to prolonged inflammation of the injury site. In contrast, zebrafish show excellent repair of nerve connections after spinal injury and this is associated with controlling inflammation. Due to recent advances in genetic technology (CRISPR/Cas9) we can now determine the function of genes that influence regeneration in the living zebrafish in a matter of days. Here we devise a very rapid screening method for the function of inflammation-related genes in zebrafish larvae after spinal cord injury. We find a number of genes that are necessary for repair of nerve connections and control of the inflammation after injury. This provides important leads to improve our understanding of the role of inflammation in spinal cord injury. Moreover, our fast and robust screening method can be adopted by other researchers to screen for gene functions in a whole animal, which was previously not easily possible.