Apr 4, 2020

Phenotypic screening using synthetic CRISPR gRNAs reveals pro-regenerative genes in spinal cord injury

BioRxiv : the Preprint Server for Biology
Marcus KeatingeThomas Becker

Abstract

Acute CRISPR/Cas9 targeting offers the opportunity for scalable phenotypic genetic screening in zebrafish. However, the unpredictable efficiency of CRISPR gRNA (CrRNA) activity is a limiting factor. Here we describe how to resolve this by pre-screening CrRNAs for high activity in vivo, using a simple standardised assay based on restriction fragment length polymorphism analysis (RFLP). We targeted 350 genomic sites with synthetic RNA Oligo guide RNAs (sCrRNAs) in zebrafish embryos and found that almost half exhibited > 90% efficiency in our RFLP assay. Having the ability to preselect highly active sCrRNAs (haCRs), we carried out a focussed phenotypic screen of 30 macrophage-related genes in spinal cord regeneration and found 10 genes whose disruption impaired axonal regeneration. Four (tgfb1a, tgfb3, tnfa, sparc) out of 5 stable mutants subsequently analysed retained the acute haCR phenotype, validating the efficiency of this approach. Mechanistically, lack of tgfb1a leads to a prolonged immune response after injury, which inhibits regeneration. Our rapid and scalable screening approach has identified functional regulators of spinal cord regeneration, and can be applied to study any biological function of interest.

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Mentioned in this Paper

Proteasome Pathway
MRNA Maturation
Protein Degradation, Metabolic
Biologic Development
Trafficking
Red fluorescent protein
Fluorescent stain
Tandem
Green Fluorescent Proteins
Proteasome Complex Location (Sensu Eukarya)

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