InDelphi-mESC prediction, indel, out-of-frame indel and in-frame rates for the different crispants, determined by NGS from pools of 1dpf embryos. The percentage of reads likely to affect the protein function is calculated based on the percentage of reads with out-of-frame indels combined with the percentage of reads with in-frame deletions of equal to or more than 6 basepairs.

Measurements of osteoblast-positive surface area and mineralized surface area of the head skeleton in 7 and 14 dpf crispants. (a) Schematic overview of the ventral and lateral perspective of the head of zebrafish larvae at 7 and 14 dpf. The surface area of the total head is circled with a dashed line. Arrows point to the notochord tip (n), the operculum (o) and the mineralized vertebrae. The eyes (e) and parasphenoid (p) are also shown. (b) Quantification of the osteoblast-positive surface area of the total head and the mineralized surface area of the total head and notochord tip at 7 dpf in comparison with their respective controls. (c) Quantification of the osteoblast-positive surface area of the total head and the mineralized surface area of the total head, operculum and notochord tip and number of vertebrae at 14 dpf in comparison with their respective controls. For easier visualization, obtained results were normalized to the respective controls (normalization = individual value crispant (or control) / mean control) (n=10). Statistical significance is evaluated using the unpaired T-test on non-normalized data and significant differences were visualized using an asterix (* = p ≤ 0,05; ** = p ≤ 0,01; *** = p ≤ 0,001; **** = p ≤ 0,0001). Error bars show the standard deviation of non-normalized data.

RT-qPCR expression analysis of runx2, sp7, bglap and col1a1a in crispants at 7 (a) and 14 dpf (b) and their respective controls, normalized according to the controls (normalization = individual values crispant (or control) / mean control). Statistical significance is evaluated using the unpaired T-test on non-normalized data and significant differences were visualized using an asterix (* = p ≤ 0,05; ** = p ≤ 0,01; *** = p ≤ 0,001; **** = p ≤ 0,0001). Error bars show the standard deviation of non-normalized data.

ARS images and survival analysis of mbtps2 crispants. (a) ARS images of control fish and crispants for mbtps2, showing severe craniofacial abnormalities (arrows). (b) Survival curve of controls and crispants for mbtps2, showing a reduction in survival starting from 17 dpf. (c) Survival curve of controls and crispants for aldh7a1, showing a reduction in survival starting from 7 dpf.

Skeletal phenotyping of adult crispants and their respective controls (a) Pictures of ARS-stained vertebral column of a control and three crispants (from left to right: esr1, ifitm5 and creb3l1), showing fractures (arrow), fusions and compressions (squared) and malformations in the arches (arrowhead). (b) Measurements of the standard length, the head size and the eye diameter of the crispants compared to their control. The standard length was measured from the snout tip to the tail base. The head size was measured from the snout tip to the supraoccipital bone and the eye diameter was measured from the lateral ethmoid to the hyomandibula. The data was normalized for easier visualization (normalization = individual value crispants (or controls) / mean control) (n=5)). (c) Quantification of the number of fractures, fusions or compressions and malformations in the arches of crispants and the controls. For this quantification, twelve vertebrae were selected per fish and a total of 5 crispants per assay was evaluated. Statistical analysis was evaluated using the unpaired T-test on non-normalized data and significant differences were visualized using an asterix (* = p ≤ 0,05; ** = p ≤ 0,01; *** = p ≤ 0,001; **** = p ≤ 0,0001). Error bars showed the standard deviation of non-normalized data. (d) Quantification of skeletal parameters by quantitative micro-computed tomography (μCT) analysis. In the graphical representation, the different crispants were listed. Statistically significant differences from the control values for a given crispant were depicted as red bars. Significance levels were determined through the global test analysis (Supplementary Figure 7).

Overview of the different measurements in crispants at 7, 14 and 90 dpf. (-): downregulation or less, (+) upregulation or more, ns: not significant, asterix defines statistical significance (* = p ≤ 0,05; ** = p ≤ 0,01; *** = p ≤ 0,001; **** = p ≤ 0,0001).