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.
Highlights
Zebrafish, in contrast to mammals, functionally regenerate axonal connections across the injury site after spinal cord injury
Nerve connections that are severed in spinal cord injury do not heal, which can lead to permanent paralysis
Due to recent advances in genetic technology (CRISPR/Cas9) we can determine the function of genes that
Summary
In contrast to mammals, functionally regenerate axonal connections across the injury site after spinal cord injury. Previous work has shown that the presence of blood-derived macrophages is crucial for axonal reconnection in the injured larval spinal cord and recovery from paralysis [3] These macrophages control the injury site environment by reducing the number of anti-regenerative neutrophils and by mitigating the injury-induced expression of pro-inflammatory cytokines, such as il1b by neutrophils and other cell types. For robust and quick pre-screening, we used a restriction fragment length polymorphism (RFLP) —based approach in which the sCrRNA cas cut site overlaps with restriction enzyme recognition sites This ensures that we retain a high degree of freedom in target selection and scalability of the approach
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