A: Example gels to assess sCrRNA in vivo mutation rate by resistance to restriction enzyme digest (RFLP) are shown. These indicate > 90% mutation rate (top), medium mutation rate (middle) and no detectable mutation rate (bottom). Each lane is derived from one animal. The chart shows the distribution of > 350 sCrRNAs by in vivo mutation rate. B-D: Activities of individual sCrRNAs show weak correlation with predicted in silico efficiency using different prediction rules (see results). E: sCrRNA activities are not correlated with their relative position to the start codon in vivo.

A: A schematic representation of the spinal cord regeneration assay. Percentages indicate expected proportions of larvae with an injury site bridged by axons in controls. B: Example images of unlesioned, non-bridged (star indicates gap of neuronal labeling) and bridged spinal cord (white arrow) are shown (lateral views). Scale bar = 50 μm. C: Results of spinal cord regeneration screen for all screened genes at 48 hpl are shown. Significant reductions in bridging, normalised to control lesioned animals, are observed for cst7 (p < 0.0001), sparc (p = 0.04), tgfb1a (p = 0.03), tgfb3 (p = 0.005), tnfa (p < 0.0001), ifngr1 (p = 0.0013,) hspd1 (p = 0.011), tbrg1 (p = 0.0494, serpinb1 (p = 0.0279), and mertk (p = 0.0195); * indicates significance at 48 hpl; # indicates significance at 24 hpl (see S2 Fig); number of larvae per experiment are indicated at the bottom of each bar. For dpm3 no viable larvae could be raised. A single sCrRNA targeting a key functional domain was used to target ctsd, abca7, sparc, clip3, abca1b, tnfa, tgfb1a and tgfb3. Two sCrRNAs were used to target all remaining genes. D: Mutant analysis confirms axonal phenotypes for sparc (p = 0.0189), tgfb1a (p = 0.019), tgfb3 (p = 0.043) and tnfa (p = 0.024), but not for cst7 (p = 0.079) at 48 hpl. The table compares the magnitude of effects between acute injection and in mutants. Fischer’s exact test was used for all analyses.

A: Lateral views of lesion sites in larval zebrafish are shown with the indicated markers and experimental conditions at 48 hpl. B-C: Quantifications show that numbers of macrophages were not altered by injecting any of the indicated haCRs (B; one-way ANOVA with Bonferroni’s multiple comparison test; Theoretical power = 0.85 to see a similar increase as for neutrophils in C), neutrophils were increased in number in tgf1b haCRs injected animals (one-way ANOVA with Bonferroni’s multiple comparison test, P = 0.0006), but not in tgfb3 haCRs injected animals (p = 0.32). D: Animals injected with tgfb1a haCRs, but not those injected with tgfb3 haCRs (P = 0.36), displayed marked increases in il1b expression levels in the lesion site compared to lesioned controls at 48 hpl (one-way ANOVA with Bonferroni’s multiple comparison test, P = 0.0211). All transcript levels were normalized to uninjected, unlesioned controls. E-F:tgfb1a heterozygous (one-way ANOVA with Tukey’s multiple comparison test, P = 0.0497) and homozygous mutant animals (P = 0.039) show increased numbers of neutrophils, comparable to haCR-injected animals. G: Inhibition of Il-1β with YVAD rescued axonal bridging compared to the DMSO-treated control group in animals injected with tgfb1a haCRs (Fisher’s exact test * p <0.05, ** p<0.01). Numbers in B, C, F indicate numbers of animals; in D numbers of independent experiments. Error bars represent standard error of the mean (SEM). Scale bars = 50 μm.