QIAxcel system, Sanger sequencing and HRM analysis results. A) Gel image produced by QIAxcel system revealing wild type aldh7a1 sequence, as well as the heterozygous and homozygous 5bp deletion. The DNA sequences shown in the lower panel reveal the complement reverse sequences. The broken line indicates the nucleotides that were deleted by CRISPR/Cas9 in the homozygous fish. B) The HRM melting plots are displayed. The heterozygous sequence is identified by the double peak. The homozygous sequence is identified by a peak at 76.5 degrees Celsius, and the wild type sequence is identified by a peak at 77.5 degrees Celsius.

Daily survival of heterozygous 5 bp deletion male and female zebrafish embryos. Y axis to the left indicates total number of embryos in the tank. Y axis to the right indicates the number of dead embryos genotyped and presented as a histogram. X axis shows dpf.

Locomotor behavior of zebrafish embryos using Viewpoint Videotrack for ZebrafishTM. A) This represents cumulative plots of the position and velocity of a single knock-out homozygous and heterozygous 5 bp deletion aldh7a1 embryo and wild type embryo at 10 dpf during one hour of behavioral recording. B) This represents average of total time spent moving (seconds), average of total distance travelled and average of velocity for homozygous (n = 20), heterozygous (n = 20) and wild type (n = 11) embryo at 10 dpf during one hour of behavioral recording. Embryos were placed in individual wells of a flat-bottom 96-well plate and acclimated to the recording chamber before tracking began. One hour of movement data were collected. Data shown are sums of 60-minute (average ± standard error). Error bars shows standard error of the mean (SEM). * is confidence level in comparison with wild type: * is p<0.05, *** is p<0.001. † is confidence level in comparison with heterozygous 10 dpf († - p<0.05, ††† - p<0.001).

EEG results of knock-out homozygous aldh7a1 embryos and wild type embryos at 9 dpf. A) This shows spike discharges in a homozygous with no response to 100 μM diazepam, but almost normalization of EEG on 25 mM pyridoxine compared to wild type. B) This shows another homozygous with no response to phenobarbital, but almost normalization of EEG on pyridoxine (25 mM) compared to wild type. C) This shows normal EEG with spikes on pentylenetetrazole, and normalization of EEG on diazepam.

EEG results of two knock-out homozygous aldh7a1 embryos and wild type embryo at 9 dpf. The results represented as number of events (per 300 seconds), duration of each event (seconds) and amplitude (mV) of each event as baseline and on treatment. Each event was considered as a single spike discharge of the EEG recording. Right panel reports events in a knock-out homozygous aldh7a1 embryo, their treatment with 100 μM diazepam and followed by 25 mM pyridoxine treatment. The middle panel shows events in another knock-out homozygous aldh7a1 embryo, their treatment with 100 μM diazepam and followed by 25 mM pyridoxine treatment. The left panel reports events in a wild type on 15 mM pentylenetetrazole to produce spikes and treat them with 100 μM diazepam. Error bars of are standard error of the mean (SEM).

Metabolite measurement of alpha-AASA, P6C and PA. 3dpf zebrafish embryo lysates (n = 15) were analyzed by liquid chromatography tandem mass spectrometry for homozygous and heterozygous for 5 bp deletion aldh7a1 embryos and wild-type embryos. These show elevated levels of alpha-AASA, P6C and PA compared to wild type.

Western blot. Lysates of knock-out homozygous aldh7a1 zebrafish embryos (at 8 dpf) western blot analysis showing no aldh7a1 protein compared to wild-type.