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

PLoS Genetics, 2021 · DOI: https://doi.org/10.1371/journal.pgen.1009515 · Published: April 29, 2021

Neurology Genetics Regenerative Medicine & Stem Cells

Simple Explanation

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. We find a number of genes that are necessary for repair of nerve connections and control of the inflammation after injury.

Study Duration

Not specified

Participants

Zebrafish larvae

Evidence Level

In vivo phenotypic screen

Key Findings

1
Rapid screening method identifies genes necessary for nerve connection repair and inflammation control after spinal cord injury in zebrafish.
2
The study validates four genes (tgfb1a, tgfb3, tnfa, sparc) as positive regulators of successful regeneration in larval zebrafish using stable mutants.
3
Deleting tgfb1a led to prolonged presence of neutrophils and increased il1b expression, similar to effects of genetic removal of macrophages.

Research Summary

The study establishes an efficient sCrRNA screening paradigm that involves a pre-screening step to compensate for inherent variability in sCrRNA activity and to identify highly active guides (haCRs) for in vivo phenotypic screens. It uses haCRs to target 30 potentially macrophage-associated genes in a spinal cord injury assay and validates four hits through generation of stable mutant lines as playing key roles in successful spinal cord regeneration in zebrafish. The research further identifies tgfb1a as a regulator of post-injury inflammation, providing a mechanistic basis to understand how inflammation is rapidly resolved to promote recovery.

Practical Implications

Drug Development

Identified genes, particularly tgfb1a, offer potential therapeutic targets for promoting spinal cord regeneration by modulating inflammation.

Understanding Regeneration

The study enhances our understanding of the pro-regenerative role of macrophages and the immune system in zebrafish spinal cord injury, providing insights that may be applicable to mammals.

Screening Methodology

The rapid sCrRNA design and pre-selection process presented in the study can be adopted for phenotypic screening in vivo for various biological contexts.

Study Limitations

1
Phenotypic screening of regeneration with haCRs is limited to genes with no essential developmental function.
2
The study acknowledges the possibility of false negative findings resulting from functional protein still being produced despite premature STOP codons being introduced.
3
The authors cannot exclude that off-target effects exaggerated the phenotype.

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