A new rapid screening approach uses CRISPR / Cas9 technology to identify genes related to the immune system that play a crucial role in repairing spinal cord damage in zebrafish. Marcus Keatinge and Themistoklis Tsarouchas from the University of Edinburgh, UK, and their colleagues present these findings in the open access journal PLOS Genetics.
In humans and other mammals, severed nerve connections in the spinal cord do not heal, so spinal cord injury can lead to permanent paralysis. In contrast, zebrafish are able to recover from spinal cord injury in a process that involves inflammation controlled by macrophages – a type of cell in the immune system. However, the precise process by which macrophages help regenerate the spinal cord in zebrafish remains a mystery.
To help clarify this process, Keatinge, Tsarouchas, and colleagues developed a new method to quickly identify genes related to macrophages involved in zebrafish spinal cord regeneration. The strategy uses CRISPR / Cas9 technology, which allows researchers to target and disrupt specific genes, revealing their function. Molecules called synthetic RNA Oligo CRISPR RNA guides (sCrRNA) allow this specific targeting of the gene.
The researchers applied the new method to study spinal cord regeneration in zebrafish larvae. Key to the method was a screening step in which they tested over 350 sCrRNAs that target genes already known to potentially play important roles in inflammation-linked spinal cord regeneration. Introducing these sCrRNAs to zebrafish allowed the identification of 10 genes which, when disrupted, impaired recovery from spinal cord injury.
Further analysis narrowed the list down to four genes that appear to be crucial for repairing broken spinal nerve connections, validating the new method. One gene in particular, tgfb1, appears to play an essential signaling role in controlling inflammation during the recovery process.
The new method and the results could help deepen the understanding of spinal cord regeneration in zebrafish. The researchers also say the method could be adapted to search for genes that also play important roles in other biological processes.
The authors add: “Zebrafish can completely regenerate their spinal cord after injury. Using a new, very rapid screening platform, we are uncovering genes in the immune system that are essential for regeneration. We are considering our results to lead to new knowledge about the inability of mammals to regenerate and our versatile screening platform to be adapted to other disease or injury models in zebrafish. “
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