CRISPR/Cas9 dre-ca8 F0 knockout impairs early motor function, but does not significantly affect motor neuron development. (A) The mean duration of spontaneous coiling activity was reduced in ca8 crispant embryos versus Cas9 controls. Significance determined by one-way analysis of variance (ANOVA) using a Kruskal-Wallis test. (B,C) Touch-evoked response assays demonstrated significant abnormal motor function in dre-ca8 crispant versus Cas9-injected controls. Significance determined by Welch's t test. (D) dre-ca8 F0 crispants do not present obvious motoneuron development defects. Transgenic line tg(MN:GFP) expressing eGFP in motoneurons. (E) Quantification of CaP motoneuron axonal length demonstrates absence of gross abnormal defects at 30 hpf and beyond. White number highlights the position of the 7 CaP motoneurons evaluated in each animal. Significance was determined by two-way ANOVA using Sidak's multiple comparison test. (F) No difference in heart rate was observed between ca8 crispant and Cas9 control embryos at 4 dpf, demonstrating absence of gross abnormality. Significance determined by Welch's ANOVA. *P < 0.05, **P < 0.01, ****P < 0.0001, ns, not significant. hpf, hours post fertilization; dpf, days post fertilization. [Color figure can be viewed at wileyonlinelibrary.com]
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