NEW YORK – Researchers at the University of Edinburgh have identified functional regulators of spinal cord regeneration in zebrafish using synthetic RNA Oligo CRISPR guide RNAs (sCrRNAs). This new screening method, they noted, is scalable and can be applied to study any biological function of interest.
In a paper published on Thursday in PLOS Genetics, the researchers noted that zebrafish usually regenerate robustly after spinal cord injuries, and that this regeneration is promoted by macrophages that control post-injury inflammation. However, how the macrophages regulate this process is poorly understood.
In order to address this issue, they conducted a rapid in vivo phenotypic screen for macrophage-related genes that promote regeneration after spinal injury. Specifically, they injected sCrRNAs that were prescreened for high activity in vivo in the general context of nervous tissue injury or disease. They rapidly identified that about half of 350 prescreened sCrRNAs were highly active, with an efficiency of more than 90 percent.
"Synthetic gRNAs are much better in causing somatic mutations than traditional ones, but not all them are equally efficient," senior author Thomas Becker said in an email. "To make full use of synthetic gRNAs, we introduced an easy prescreening step in vivo to be confident that genes are actually disrupted in functional screens."
They went on to use these highly active guide RNAs (termed haCRs) to effectively target 30 potentially macrophage-related genes. In a subsequent analysis, the researchers found that disruption of 10 of these genes impaired axonal regeneration following spinal cord injury. They further narrowed this list down to five genes that seemed essential for regeneration from an early time point and generated stable mutants using haCRs. Four of these mutants (tgfb1a, tgfb3, tnfa, and sparc) retained the acute haCR phenotype.
Mechanistically, they found, deleting tgfb1a led to a prolonged presence of neutrophils and increased Il-1beta expression, and the zebrafish failed to resolve post-injury inflammation. Therefore, they concluded that tgfb1a encodes a signaling molecule that controls inflammation after spinal injury in zebrafish, providing a mechanistic understanding of the pro-regenerative role of their immune system.
The screen also found additional regeneration-relevant genes that were confirmed in zebrafish mutants. For example, the researchers said, Tnfa is mainly produced by macrophages in the spinal injury site and has previously been shown to be necessary for larval spinal cord regeneration. They confirmed these findings in this study using a stable tnfa mutant.
"Finding functional immune system-related genes in successful spinal cord regeneration was possible due to our novel screening paradigm, facilitated by in vivo prescreening of sCrRNAs for activity," the authors wrote.
They also highlighted the screen's relatively high hit rate of 33 percent, though they noted that this was expected, as the genes were preselected for likely functions in macrophages that play a crucial role in controlling the inflammation in successful spinal cord regeneration in zebrafish. They further confirmed a role for four of five stable mutants in spinal cord regeneration.
"This indicates that the screening paradigm has a relatively low rate of false positive findings," the authors wrote, but they also cautioned that they encountered a false positive in cst7 and that two of the other four mutants showed a weaker phenotype than after acute injection of haCRs.