Fig. 6.

CRISPR/Cas9-mediated disruption of col12a1a/b leads to depletion of mRNA and impaired axonal regeneration. a Cartoon showing strategy used for CRISPR/Cas9-mediated col12a1a/b gene disruption. cas9 mRNA and small guide-RNA (gRNA) targeting col12a1a and col12a1b were co-injected into the zygote. Crispants were analysed for col12a1a/b mRNA levels and axonal regeneration as indicated in the timeline. b Restriction fragment length polymorphisms (RFLP) analysis reveals efficient somatic mutation in the gRNA target site (indicated by resistance to restriction endonuclease digest; arrowheads) in both col12a1 paralogs after microinjection. c col12a1a/b Crispants exhibit reduced developmental col12a1a and col12a1b mRNA expression, determined by quantitative RT-PCR. Representative plot from three independent experiments for each mRNA are shown. mRNA levels in gfp gRNA-injected control animals were set to 100%. d col12a1a/b Crispants exhibit reduced col12a1a and col12a1b mRNA levels after lesion, determined by quantification of fluorescent in situ hybridization signal in the lesion site (t-test: **P < 0.01, *P < 0.05). e col12a1a/b Crispants show reduced axonal bridging events after lesion (t-test: **P < 0.01). a–e Views are lateral (dorsal is up, rostral is left). BF: brightfield. Scale bars: 200 µm e and 100 µm d,e. Error bars indicate s.e.m