RT Journal Article
SR Electronic
T1 Phenotypic screening using synthetic CRISPR gRNAs reveals pro-regenerative genes in spinal cord injury
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2020.04.03.023119
DO 10.1101/2020.04.03.023119
A1 Marcus Keatinge
A1 Themistoklis M. Tsarouchas
A1 Tahimina Munir
A1 Juan Larraz
A1 Davide Gianni
A1 Hui-Hsin Tsai
A1 Catherina G. Becker
A1 David A. Lyons
A1 Thomas Becker
YR 2020
UL http://biorxiv.org/content/early/2020/04/04/2020.04.03.023119.abstract
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 inflammation