PT  - JOURNAL ARTICLE
AU  - Marcus Keatinge
AU  - Themistoklis M. Tsarouchas
AU  - Tahimina Munir
AU  - Juan Larraz
AU  - Davide Gianni
AU  - Hui-Hsin Tsai
AU  - Catherina G. Becker
AU  - David A. Lyons
AU  - Thomas Becker
TI  - Phenotypic screening using synthetic CRISPR gRNAs reveals pro-regenerative genes in spinal cord injury
AID  - 10.1101/2020.04.03.023119
DP  - 2020 Jan 01
TA  - bioRxiv
PG  - 2020.04.03.023119
4099  - http://biorxiv.org/content/early/2020/05/05/2020.04.03.023119.short
4100  - http://biorxiv.org/content/early/2020/05/05/2020.04.03.023119.full
AB  - 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 prescreening 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.HIGHLIGHTS- Synthetic CRISPR gRNAs are highly active- in vivo pre-screening allows rapid assessment of CRISPR gRNA activity- Phenotypic CRISPR screen reveals crucial genes for spinal cord regeneration- tgfb1a promotes spinal regeneration by controlling inflammationCompeting Interest StatementThe authors have declared no competing interest.