otp crispants have disrupted myelin. a, b An example of Sanger sequencing shows that otpa and otpb genes were disrupted after injection with CRISPR/Cas9. The sgRNA sequence is underlined in red. c Percentage of in-frame and out-of-frame mutations from otpa and otpb PCR amplicons (all PCR products have mutations). d Representative sequences from individually cloned PCR products. The sgRNA sequence is underlined in red. Insertions and deletions are shown as red letters and dashes, respectively. e, fTg (mbp:egfp) at 72hpf by confocal microscopy imaging. e control. fotp crispant. g h Quantification of myelin deficits. N = 12 for the control group, and N = 15 for the otp crispant group. g Percentage of myelin formation along the tract (unpaired t test, p = 0.0001). Two-headed arrows (e, f) show the intact myelin. The lengths of intact myelin are added and the percentage is calculated by dividing by the length of the entire image window. The same threshold is set in each z projection for each embryo, and the length is calculated for visible segments. h Thickness of myelin sheaths (unpaired t test, p = 0.0045). The red box (e, f) is drawn around the visible myelin and the height of the box was used to calculate the thickness of the myelin sheaths. i ELISA shows that the dopamine levels (ng dopamine/larvae weight) are decreased in otp crispants (unpaired t test, p = 0.0079)
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