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

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 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.

PTPN21/Pez Is a Novel and Evolutionarily Conserved Key Regulator of Inflammation In Vivo

“…To investigate what effects the loss of PTPN21 and MEGF10 have on the development of zebrafish leukocytes, we independently utilized the transgenic neutrophil line Tg(lysC:NLS-mScarlet) 23 and macrophage reporter lines Tg(mpeg1.1:NLS-mScarlet) and Tg ( mpeg1.1:EGFP ) to generate CRISPR-Cas9-mediated mutant larvae (“crispants”; Figures S2 N and S2P). Using restriction fragment length polymorphism analysis (RFLP), 24 we were able to validate the successful generation of F0 crispant larvae ( Figures S2 O and S2Q). Imaging the entirety of the crispant fish revealed leukocyte distribution was unaltered when compared with wild type ( Figure 4 A).…”

“…CRISPR/Cas9-mediated mutant larvae “Crispants” were generated as described previously 24 . Briefly, CRISPR guide RNA (crRNA) sequences in which restriction enzyme recognition sequences overlapped the Cas9 cut site were identified in PTPN21 and MEGF10 exons and commercially synthesized (Sigma-Aldrich).…”

“…For macrophage experiments wound recruitment was compared to uninjected clutch-mates. Genotyping to confirm successful gene editing was performed following DNA extraction from individual larvae (95°C in 50mM NaOH for 1 hr, followed by addition of 0.5 M Tris-HCl pH 8.0) as previously described 24 ). PCR of the edited region was performed using MyTaq Red Mix (Meridian Bioscience) and fragments were subsequently digested over night by the addition of 1 μL BslI or MwoI (NEBiolabs) directly to the reaction.…”