Clinical images of the proband. A Distinct facial features of the proband, including midfacial hypoplasia, prominent forehead, long face, almond-shaped eyes, short nose, long philtrum, thin upper lip, large and protruding ears, and sparse teeth, without obvious short and stubby fingers, deformities in the limbs or joint contractures. B Growth curve chart for the proband’s height/length. C Growth curve chart for the proband’s weight. D Growth curve chart for the proband’s head circumference

Auxiliary examination of the proband. A The peripheral blood flow cytometry results of the proband indicated a significant decrease in CD107a in the NK cells. B Video electroencephalogram revealed that the proband had abnormal discharges. In the awake state, moderate theta activity was detected at 4–7 Hz in the background without an occipital dominant rhythm. During wakefulness and sleep, spike-slow wave or slow wave bursts were observed in the left parietal and posterior temporal regions, which spread diffusely. C Brain magnetic resonance imaging (MRI) revealed an arachnoid cyst in the occipital region, along with bilateral enlarged lateral ventricles. D Spinal X-rays showing mild scoliosis with a Cobb angle of 4°, as indicated by the arrows. Limb X-rays revealed no abnormalities. E The Sanger sequencing of trios

Auxiliary examination of the proband. A The peripheral blood flow cytometry results of the proband indicated a significant decrease in CD107a in the NK cells. B Video electroencephalogram revealed that the proband had abnormal discharges. In the awake state, moderate theta activity was detected at 4–7 Hz in the background without an occipital dominant rhythm. During wakefulness and sleep, spike-slow wave or slow wave bursts were observed in the left parietal and posterior temporal regions, which spread diffusely. C Brain magnetic resonance imaging (MRI) revealed an arachnoid cyst in the occipital region, along with bilateral enlarged lateral ventricles. D Spinal X-rays showing mild scoliosis with a Cobb angle of 4°, as indicated by the arrows. Limb X-rays revealed no abnormalities. E The Sanger sequencing of trios

Characteristics of the variant and protein levels of STT3A. A Conservation analysis of this variant, which is conserved across multiple species. B Molecular modeling of the variant indicated a reduction in hydrogen bonds around the residue. C Molecular modeling of STT3A showing that the heterozygous missense variant is located within the binding site. D Western blot analysis of whole-cell protein extracts from transfected HEK293T cells. Compared with that in the WT group, the STT3A expression level in the Asp167Tyr group was significantly lower

The phenotype validation of CRISPR-Cas9 established heterozygous knockdown zebrafish. A Bright-field images of the Cas9 control group and the stt3a crispant group zebrafish. B Fluorescence images of the brains of the zebrafish in the Cas9 control group and the stt3a crispant group. C, D Measurements of eye distance and body length in the Cas9 control group (n = 19 fish) vs. the stt3a crispant group (n = 29 fish; unpaired t test). E–I Measurements of the eye distance-to-body length ratio, forebrain area, midbrain area, hindbrain area, and total brain area in the Cas9 control group (n = 19 fish) vs. the stt3a crispant group (n = 29 fish; unpaired t test). J Representative fluorescence images of the lower jaw cartilage morphology of the zebrafish in the Cas9 control group and the stt3a crispant group. K–I Measurements of basal length and Ceratohyal angle in the Cas9 control group (n = 21 fish) vs. the stt3a crispant group (n = 20 fish; unpaired t test). M–O Measurements of Meckel’s length, Palatoquadrant’s length, and Ceratohyal’s length in the Cas9 control group (n = 21 fish) vs. the stt3a crispant group (n = 20 fish; unpaired t test). P–Q Representative images of zebrafish bone staining in the Cas9 control group and the stt3a crispant. Top view and side view. R Number of mineralized bones in the Cas9 control group (n = 8 fish) vs. the stt3a crispant group (n = 8 fish; unpaired t test). S Plot of the movement distance of the Cas9 control group (n = 39 fish) and stt3a crispant group (n = 39 fish) as a line graph with 1 min intervals; the error bars represent the standard error of the mean (SEM). T, U Measurements of movement distance and maximum speed in the Cas9 control group (n = 39 fish) vs. the stt3a crispant group (n = 39 fish; unpaired t test). V A 15 min trajectory plot where the color of the movement trajectory represents the speed, allowing observation that the Cas9 control group and stt3a crispant group exhibit similar trajectory densities and proportions of high-speed movement trajectories. (W) When ≥ 40 mm/s is used as the high-speed screening threshold to generate a 15 min heatmap of high-speed movement (with different colors representing frequency values), the proportions of individuals exhibiting high-speed movement and the frequency of high-speed movement occurrence are similar between the Cas9 control group and the stt3a crispant group. (X) Processing the movement distance data of the Cas9 control group (n = 39) and the stt3a crispant group (n = 38) into line graphs with 30 s intervals; the error bars represent the SEM. Y, Z L means light, D means dark. As the response of zebrafish larvae to changes in light and darkness was not significant in the first cycle, it was not included in the analysis. Data from cycles 2–4 were analyzed by averaging the three points corresponding to each time point in the three cycles. Measurements of movement distance and maximum speed in the Cas9 control group (n = 39 fish) vs. the stt3a crispant group (n = 38 fish; unpaired t test) under light and dark stimuli

The phenotype validation of CRISPR-Cas9 established heterozygous knockdown zebrafish. A Bright-field images of the Cas9 control group and the stt3a crispant group zebrafish. B Fluorescence images of the brains of the zebrafish in the Cas9 control group and the stt3a crispant group. C, D Measurements of eye distance and body length in the Cas9 control group (n = 19 fish) vs. the stt3a crispant group (n = 29 fish; unpaired t test). E–I Measurements of the eye distance-to-body length ratio, forebrain area, midbrain area, hindbrain area, and total brain area in the Cas9 control group (n = 19 fish) vs. the stt3a crispant group (n = 29 fish; unpaired t test). J Representative fluorescence images of the lower jaw cartilage morphology of the zebrafish in the Cas9 control group and the stt3a crispant group. K–I Measurements of basal length and Ceratohyal angle in the Cas9 control group (n = 21 fish) vs. the stt3a crispant group (n = 20 fish; unpaired t test). M–O Measurements of Meckel’s length, Palatoquadrant’s length, and Ceratohyal’s length in the Cas9 control group (n = 21 fish) vs. the stt3a crispant group (n = 20 fish; unpaired t test). P–Q Representative images of zebrafish bone staining in the Cas9 control group and the stt3a crispant. Top view and side view. R Number of mineralized bones in the Cas9 control group (n = 8 fish) vs. the stt3a crispant group (n = 8 fish; unpaired t test). S Plot of the movement distance of the Cas9 control group (n = 39 fish) and stt3a crispant group (n = 39 fish) as a line graph with 1 min intervals; the error bars represent the standard error of the mean (SEM). T, U Measurements of movement distance and maximum speed in the Cas9 control group (n = 39 fish) vs. the stt3a crispant group (n = 39 fish; unpaired t test). V A 15 min trajectory plot where the color of the movement trajectory represents the speed, allowing observation that the Cas9 control group and stt3a crispant group exhibit similar trajectory densities and proportions of high-speed movement trajectories. (W) When ≥ 40 mm/s is used as the high-speed screening threshold to generate a 15 min heatmap of high-speed movement (with different colors representing frequency values), the proportions of individuals exhibiting high-speed movement and the frequency of high-speed movement occurrence are similar between the Cas9 control group and the stt3a crispant group. (X) Processing the movement distance data of the Cas9 control group (n = 39) and the stt3a crispant group (n = 38) into line graphs with 30 s intervals; the error bars represent the SEM. Y, Z L means light, D means dark. As the response of zebrafish larvae to changes in light and darkness was not significant in the first cycle, it was not included in the analysis. Data from cycles 2–4 were analyzed by averaging the three points corresponding to each time point in the three cycles. Measurements of movement distance and maximum speed in the Cas9 control group (n = 39 fish) vs. the stt3a crispant group (n = 38 fish; unpaired t test) under light and dark stimuli

Representative electrophysiological signals of zebrafish in the Cas9 control group and the stt3a crispant group. Number of abnormally discharged zebrafish in the Cas9 control group (n = 30 fish) vs. the stt3 crispant group (n = 29 fish; chi-square test)